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BackgroundThe Coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of SARS-CoV-2 infections has decreased significantly; the long-term outlook of COVID-19 remains a serious cause of high death worldwide; with the mortality rate still surpassing even the worst mortality rates recorded for the influenza viruses. The continuous emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, have prolonged the COVID-19 pandemic and outlines the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs.MethodsIn the present study, we designed a multi-epitope-based Coronavirus vaccine that incorporated B, CD4+, and CD8+T cell epitopes conserved among all known SARS-CoV-2 VOCs and selectively recognized by CD8+and CD4+T-cells from asymptomatic COVID-19 patients irrespective of VOC infection. The safety, immunogenicity, and cross-protective immunity of this pan-Coronavirus vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model.ResultsThe Pan-Coronavirus vaccine: (i) is safe; (ii) induces high frequencies of lung-resident functional CD8+and CD4+TEMand TRMcells; and (iii) provides robust protection against virus replication and COVID-19-related lung pathology and death caused by six SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma or P1 (B.1.1.28.1), Delta (lineage B.1.617.2) and Omicron (B.1.1.529).Conclusions: A multi-epitope pan-Coronavirus vaccine bearing conserved human B and T cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that cleared the virus, and reduced COVID-19-related lung pathology and death caused by multiple SARS-CoV-2 VOCs.

Four major mucosal-associated chemokines, CCL25, CCL28, CXCL14, and CXCL17, play an important role in protecting mucosal surfaces from infectious pathogens. However, their role in protection against genital herpes remains to be fully explored. The CCL28 is a chemoattractant for the CCR10 receptor–expressing immune cells and is produced homeostatically in the human vaginal mucosa (VM). In this study, we investigated the role of the CCL28/CCR10 chemokine axis in mobilizing protective antiviral B and T cell subsets into the VM site of herpes infection. We report a significant increase in the frequencies of HSV-specific memory CCR10+CD44+CD8+ T cells, expressing high levels of CCR10, in herpes-infected asymptomatic (ASYMP) women compared with symptomatic women. Similarly, a significant increase in the CCL28 chemokine (a ligand of CCR10), was detected in the VM of herpes-infected ASYMP C57BL/6 mice, associated with the mobilization of high frequencies of HSV-specific effector memory CCR10+CD44+CD62L−CD8+ TEM cells and memory CCR10+B220+CD27+ B cells in the VM of HSV-infected ASYMP mice. Inversely, compared with wild-type C57BL/6 mice, the CCL28 knockout (CCL28−/−) mice (1) appeared to be more susceptible to intravaginal infection and reinfection with HSV type 2, and (2) exhibited a significant decrease in the frequencies of HSV-specific effector memory CCR10+CD44+CD62L−CD8+ TEM cells and of memory CD27+B220+ B cells in the infected VM. These findings suggest a critical role of the CCL28/CCR10 chemokine axis in the mobilization of antiviral memory B and T cells within the VM to protect against genital herpes infection and disease.

Over the last two decades, there have been three deadly human outbreaks of coronaviruses (CoVs) caused by SARS-CoV, MERS-CoV, and SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats and transmitted to humans via various intermediate animal reservoirs. It remains highly possible that other global COVID pandemics will emerge in the coming years caused by yet another spillover of a bat-derived SARS-like coronavirus (SL-CoV) into humans. Determining the Ag and the human B cells, CD4+ and CD8+ T cell epitope landscapes that are conserved among human and animal coronaviruses should inform in the development of future pan-coronavirus vaccines. In the current study, using several immunoinformatics and sequence alignment approaches, we identified several human B cell and CD4+ and CD8+ T cell epitopes that are highly conserved in 1) greater than 81,000 SARS-CoV-2 genome sequences identified in 190 countries on six continents; 2) six circulating CoVs that caused previous human outbreaks of the common cold; 3) nine SL-CoVs isolated from bats; 4) nine SL-CoV isolated from pangolins; 5) three SL-CoVs isolated from civet cats; and 6) four MERS strains isolated from camels. Furthermore, the identified epitopes: 1) recalled B cells and CD4+ and CD8+ T cells from both COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2, and 2) induced strong B cell and T cell responses in humanized HLA-DR1/HLA-A*02:01 double-transgenic mice. The findings pave the way to develop a preemptive multiepitope pan-coronavirus vaccine to protect against past, current, and future outbreaks.

SARS-CoV-2-specific memory T cells that cross-react with common cold coronaviruses (CCCs) are present in both healthy donors and COVID-19 patients. However, whether these cross-reactive T cells play a role in COVID-19 pathogenesis versus protection remain to be fully elucidated. In this study, we characterized cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells, targeting genome-wide conserved epitopes in a cohort of 147 non-vaccinated COVID-19 patients, divided into six groups based on the degrees of disease severity. We compared the frequency, phenotype, and function of these SARS-CoV-2-specific CD4+ and CD8+ T cells between severely ill and asymptomatic COVID-19 patients and correlated this with α-CCCs and β-CCCs co-infection status. Compared with asymptomatic COVID-19 patients, the severely ill COVID-19 patients and patients with fatal outcomes: (i) Presented a broad leukocytosis and a broad CD4+ and CD8+ T cell lymphopenia; (ii) Developed low frequencies of functional IFN-γ-producing CD134+CD138+CD4+ and CD134+CD138+CD8+ T cells directed toward conserved epitopes from structural, non-structural and regulatory SARS-CoV-2 proteins; (iii) Displayed high frequencies of SARS-CoV-2-specific functionally exhausted PD-1+TIM3+TIGIT+CTLA4+CD4+ and PD-1+TIM3+TIGIT+CTLA4+CD8+ T cells; and (iv) Displayed similar frequencies of co-infections with β-CCCs strains but significantly fewer co-infections with α-CCCs strains. Interestingly, the cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcome seen in asymptomatic COVID-19 patients. Our results demonstrate that, the critically ill COVID-19 patients displayed fewer co-infection with α-CCCs strain, presented broad T cell lymphopenia and higher frequencies of cross-reactive exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells. In contrast, the asymptomatic COVID-19 patients, appeared to present more co-infections with α-CCCs strains, associated with higher frequencies of functional cross-reactive SARS-CoV-2-specific CD4+ and CD8+ T cells. These findings support the development of broadly protective, T-cell-based, multi-antigen universal pan-Coronavirus vaccines.KEY POINTSA broad lymphopenia and lower frequencies of SARS-CoV-2-specific CD4+ and CD8+ T-cells were associated with severe disease onset in COVID-19 patients.High frequencies of phenotypically and functionally exhausted SARS-CoV-2-specific CD4+ and CD8+ T cells, co-expressing multiple exhaustion markers, and targeting multiple structural, non-structural, and regulatory SARS-CoV-2 protein antigens, were detected in severely ill COVID-19 patients.Compared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented more functional cross-reactive CD4+ and CD8+ T cells targeting conserved epitopes from structural, non-structural, and regulatory SARS-CoV-2 protein antigens.The cross-reactive SARS-CoV-2 epitopes that recalled the strongest CD4+ and CD8+ T cell responses in unexposed healthy donors (HD) were the most strongly associated with better disease outcomes seen in asymptomatic COVID-19 patients.Compared to severely ill COVID-19 patients and to patients with fatal outcomes, the (non-vaccinated) asymptomatic COVID-19 patients presented higher rates of co-infection with the α-CCCs strains.Compared to patients with mild or asymptomatic COVID-19, severely ill symptomatic patients and patients with fatal outcomes had more exhausted SARS-CoV-2-speccific CD4+ and CD8+ T cells that preferentially target cross-reactive epitopes that share high identity and similarity with the β-CCCs strains.

Since mitochondrial dysfunction is implicated in the pathogenesis of AMD, this study is based on the premise that repurposing of mitochondria-stabilizing FDA-approved drugs such as PU-91, might rescue AMD RPE cells from AMD mitochondria-induced damage. The PU-91 drug upregulates PGC-1α which is a critical regulator of mitochondrial biogenesis. Herein, we tested the therapeutic potential of PU-91 drug and examined the additive effects of treatment with PU-91 and esterase inhibitors i.e., EI-12 and EI-78, using the in vitro transmitochondrial AMD cell model. This model was created by fusing platelets obtained from AMD patients with Rho0 i.e., mitochondria-deficient, ARPE-19 cell lines. The resulting AMD RPE cell lines have identical nuclei but differ in their mitochondrial DNA content, which is derived from individual AMD patients. Briefly, we report significant improvement in cell survival, mitochondrial health, and antioxidant potential in PU-91-treated AMD RPE cells compared to their untreated counterparts. In conclusion, this study identifies PU 91 as a therapeutic candidate drug for AMD and repurposing of PU-91 will be a smoother transition from lab bench to clinic since the pharmacological profiles of PU-91 have been examined already.

Over the last two decades, there have been three deadly human outbreaks of Coronaviruses (CoVs) caused by emerging zoonotic CoVs: SARS-CoV, MERS-CoV, and the latest highly transmissible and deadly SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats, the natural hosts, and transmitted to humans via various intermediate animal reservoirs. Because there is currently no universal pan-Coronavirus vaccine available, two worst-case scenarios remain highly possible: (1) SARS-CoV-2 mutates and transforms into a seasonal “flu-like” global pandemic; and/or (2) Other global COVID-like pandemics will emerge in the coming years, caused by yet another spillover of an unknown zoonotic bat-derived SARS-like Coronavirus (SL-CoV) into an unvaccinated human population. Determining the antigen and epitope landscapes that are conserved among human and animal Coronaviruses as well as the repertoire, phenotype and function of B cells and CD4 (+) and CD8 (+) T cells that correlate with resistance seen in asymptomatic COVID-19 patients should inform in the development of pan-Coronavirus vaccines (1) . In the present study, using several immuno-informatics and sequence alignment approaches, we identified several human B-cell, CD4 (+) and CD8 (+) T cell epitopes that are highly conserved in: ( i ) greater than 81,000 SARS-CoV-2 human strains identified to date in 190 countries on six continents; ( ii ) six circulating CoVs that caused previous human outbreaks of the “Common Cold”; ( iii ) five SL-CoVs isolated from bats; ( iv ) five SL-CoV isolated from pangolins; ( v ) three SL-CoVs isolated from Civet Cats; and ( vi ) four MERS strains isolated from camels. Furthermore, we identified cross-reactive asymptomatic epitopes that: ( i ) recalled B cell, CD4 (+) and CD8 (+) T cell responses from both asymptomatic COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2; and ( ii ) induced strong B cell and T cell responses in “humanized” Human Leukocyte Antigen (HLA)-DR/HLA-A*02:01 double transgenic mice. The findings herein pave the way to develop a pre-emptive multi-epitope pan-Coronavirus vaccine to protect against past, current, and potential future outbreaks.

Over the last two decades, there have been three deadly human outbreaks of Coronaviruses (CoVs) caused by SARS-CoV, MERS-CoV, and SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats and transmitted to humans via various intermediate animal reservoirs. It remains highly possible that other global COVID pandemics will emerge in the coming years, caused by yet another spillover of a bat-derived SARS-like Coronavirus (SL-CoV) into humans. Determining the antigen and the human B cells, CD4(+) and CD8(+) T cells epitope landscapes that are conserved among human and animal Coronaviruses should inform in the development of future pan-Coronavirus vaccines. In the present study, using several immuno-informatics and sequence alignment approaches, we identified several human B-cell, CD4(+) and CD8(+) T cell epitopes that are highly conserved in: (i) greater than 81,000 SARS-CoV-2 strains identified in 190 countries on six continents; (ii) six circulating CoVs that caused previous human outbreaks of the “Common Cold”; (iii) nine SL-CoVs isolated from bats; (iv) nine SL-CoV isolated from pangolins; (v) three SL-CoVs isolated from civet cats; and (vi) four MERS strains isolated from camels. Furthermore, we identified epitopes: (i) recalled B cell, CD4(+) and CD8(+) T cells from both COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2; and (ii) induced strong B cell and T cell responses in “humanized” Human Leukocyte Antigen (HLA)-DR1/HLA-A*02:01 double transgenic mice. The findings pave the way to develop a pre-emptive multi-epitope pan-Coronavirus vaccine to protect against past, current, and future outbreaks.

Over the last two decades, there have been three deadly human outbreaks of Coronaviruses (CoVs) caused by emerging zoonotic CoVs: SARS-CoV, MERS-CoV, and the latest highly transmissible and deadly SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats, the natural hosts, and transmitted to humansviavarious intermediate animal reservoirs. Because there is currently no universal pan-Coronavirus vaccine available, two worst-case scenarios remain highly possible: (1) SARS-CoV-2 mutates and transforms into a seasonal “flu-like” global pandemic; and/or (2) Other global COVID-like pandemics will emerge in the coming years, caused by yet another spillover of an unknown zoonotic bat-derived SARS-like Coronavirus (SL-CoV) into an unvaccinated human population. Determining the antigen and epitope landscapes that are conserved among human and animal Coronaviruses as well as the repertoire, phenotype and function of B cells and CD4+and CD8+T cells that correlate with resistance seen in asymptomatic COVID-19 patients should inform in the development of pan-Coronavirus vaccines1. In the present study, using several immuno-informatics and sequence alignment approaches, we identified several human B-cell, CD4+and CD8+T cell epitopes that are highly conserved in: (i) greater than 81,000 SARS-CoV-2 human strains identified to date in 190 countries on six continents; (ii) six circulating CoVs that caused previous human outbreaks of the “Common Cold”; (iii) five SL-CoVs isolated from bats; (iv) five SL-CoV isolated from pangolins; (v) three SL-CoVs isolated from Civet Cats; and (vi) four MERS strains isolated from camels. Furthermore, we identified cross-reactive asymptomatic epitopes that: (i) recalled B cell, CD4+and CD8+T cell responses from both asymptomatic COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2; and (ii) induced strong B cell and T cell responses in “humanized” Human Leukocyte Antigen (HLA)-DR/HLA-A*02:01 double transgenic mice. The findings herein pave the way to develop a pre-emptive multi-epitope pan-Coronavirus vaccine to protect against past, current, and potential future outbreaks.

Emblica officinalis Gaetrn (i.e., Phyllanthus emblica/ Indian gooseberry/ Amla) (EO) has been used extensively as a nutraceutical in several diseases since it is known to boost immunity and offers numerous health benefits such as antioxidant, anti-inflammatory, and anti-aging effects. The goal of our study was to test the hypothesis that EO will rescue human AMD RPE transmitochondrial cells from mitochondria-induced cellular damage. AMD RPE transmitochondrial cell lines were created by fusion of mitochondria DNA-deficient APRE-19 (Rho0) cells with platelets isolated from AMD patients, and therefore had identical nuclei but differed in mitochondrial DNA content. These AMD RPE cells were treated with EO extract followed by characterization of effects of EO using cellular and molecular assays. Herein, EO significantly improved live cell number and mitochondrial membrane potential, reduced apoptosis and oxidative stress, down-regulated VEGF, and up-regulated PGC-1α. In conclusion, EO improved cellular and mitochondrial health, thereby playing a key cytoprotective role in AMD in vitro. Further studies are required to examine the mechanisms that mediate the cytoprotective effects of EO.

Author(s): Atilano, Shari R; Lee, Daniel H; Fukuhara, Paula S; Chwa, Marilyn; Nesburn, Anthony B; Udar, Nitin; Kenney, M Cristina | Abstract: PurposeTo compare the levels of gene expression for enzymes involved in production and elimination of reactive oxygen/nitrogen species (ROS/RNS) in normal human corneal cells (NL cells) with those in human corneal cells with keratoconus (KC cells) in vitro.MethodsPrimary NL and KC stromal fibroblast cultures were incubated with apocynin (an inhibitor of NADPH oxidase) or N-nitro-L-arginine (N-LLA; an inhibitor of nitric oxide synthase). ROS/RNS levels were measured using an H2 DCFDA fluorescent assay. The RT2 Profiler™ PCR Array for Oxidative Stress and Antioxidant Defense was used for initial screening of the NL and KC cultures. Transcription levels for genes related to production or elimination of ROS/RNS were analyzed using quantitative PCR. Immunohistochemistry was performed on 10 intact human corneas using antibodies against SCARA3 and CPSF3.ResultsArray screening of 84 antioxidant-related genes identified 12 genes that were differentially expressed between NL and KC cultures. Compared with NL cells, quantitative PCR showed that KC cells had decreased expression of antioxidant genes SCARA3 isoform 2 (0.59-fold, P = 0.02) and FOXM1 isoform 1 (0.61-fold, P = 0.03). KC cells also had downregulation of the antioxidant genes SOD1 (0.4-fold, P = 0.0001) and SOD3 (0.37-fold, P = 0.02) but increased expression of SOD2 (3.3-fold, P l 0.0001), PRDX6 (1.47-fold, P = 0.01), and CPSF3 (1.44-fold, P = 0.02).ConclusionThe difference in expression of antioxidant enzymes between KC and NL suggests that the oxidative stress imbalances found in KC are caused by defects in ROS/RNS removal rather than increased ROS/RNS production.

Mitochondrial-derived peptides (MDPs) are rapidly emerging therapeutic targets to combat development of neurodegenerative diseases. SHLP2 (small humanin-like peptide 2) is a newly discovered MDP that is coded from the MT-RNR2 (Mitochondrially encoded 16S rRNA) gene in mitochondrial DNA (mtDNA). In the current study, we examined the biological consequences of treatment with exogenously-added SHLP2 in an in vitro human transmitochondrial age-related macular degeneration (AMD) ARPE-19 cell model. In AMD cells, we observed significant down-regulation of the MDP-coding MT-RNR2 gene, and remarkably reduced levels of all five oxidative phosphorylation (OXPHOS) complex I-V protein subunits that are involved in the electron transport chain; these results suggested mitochondrial toxicity and abnormal OXPHOS complex protein subunits’ levels in AMD cells. However, treatment of AMD cells with SHLP2: (1) restored the normal levels of OXPHOS complex protein subunits, (2) prevented loss of viable cells and mitochondria, (3) increased the number of mtDNA copies, (4) induced anti-apoptotic effects, and (5) attenuated amyloid-β-induced cellular and mitochondrial toxicity. Cumulatively, our findings established the protective role of SHLP2 in AMD cells in vitro. In conclusion, this novel study supports the merit of SHLP2 in the treatment of AMD, a primary retinal disease that is a leading cause of blindness among the elderly population in the United States as well as worldwide.

Resveratrol is a phytoalexin, stilbenoid compound with antioxidant properties attributable to its bioactive trans-resveratrol content. This study characterized the effects of over-the-counter (OTC) resveratrol nutritional supplements and a HPLC-purified resveratrol formulation, in human transmitochondrial age-related macular degeneration (AMD) retinal pigment epithelial (RPE) patient cell lines. These cell lines, which were created by fusing blood platelets obtained from dry and wet AMD patients with mitochondria-deficient (Rho0) ARPE-19 cells, had identical nuclei (derived from ARPE-19 cells) but different mitochondria that were derived from AMD patients. After resveratrol treatment, the levels of cell viability and reactive oxygen species production were measured. Results demonstrated that treatment with different resveratrol formulations improved cell viability and decreased reactive oxygen species generation in each AMD patient cell line. Although further studies are required to establish the cytoprotective potential of resveratrol under different physiological conditions, this novel study established the positive effects of OTC resveratrol supplements in macular degeneration patient cybrid cell lines in vitro.

Over the last two decades, there have been three deadly human outbreaks of Coronaviruses (CoVs) caused by emerging zoonotic CoVs: SARS-CoV, MERS-CoV, and the latest highly transmissible and deadly SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats, the natural hosts, and transmitted to humans via various intermediate animal reservoirs. Because there is currently no universal pan-Coronavirus vaccine available, two worst-case scenarios remain highly possible: (1) SARS-CoV-2 mutates and transforms into a seasonal “flu-like” global pandemic; and/or (2) Other global COVID-like pandemics will emerge in the coming years, caused by yet another spillover of an unknown zoonotic bat-derived SARS-like Coronavirus (SL-CoV) into an unvaccinated human population. Determining the antigen and epitope landscapes that are conserved among human and animal Coronaviruses as well as the repertoire, phenotype and function of B cells and CD4+ and CD8+ T cells that correlate with resistance seen in asymptomatic COVID-19 patients should inform in the development of pan-Coronavirus vaccines. In the present study, using several immuno-informatics and sequence alignment approaches, we identified several human B-cell, CD4+ and CD8+ T cell epitopes that are highly conserved in: (i) greater than 81,000 SARS-CoV-2 human strains identified to date in 190 countries on six continents; (ii) six circulating CoVs that caused previous human outbreaks of the “Common Cold”; (iii) five SL-CoVs isolated from bats; (iv) five SL-CoV isolated from pangolins; (v) three SL-CoVs isolated from Civet Cats; and (vi) four MERS strains isolated from camels. Furthermore, we identified cross-reactive asymptomatic epitopes that: (i) recalled B cell, CD4+ and CD8+ T cell responses from both asymptomatic COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2; and (ii) induced strong B cell and T cell responses in “humanized” Human Leukocyte Antigen (HLA)-DR/HLA-A*02:01 double transgenic mice. The findings herein pave the way to develop a pre-emptive multi-epitope pan-Coronavirus vaccine to protect against past, current, and potential future outbreaks. Funding: This work is supported by the Fast-Grant PR12501 from Emergent Ventures, by a Gavin Herbert Eye Institute internal grant, by Public Health Service Research grants AI158060, AI150091, AI143348, AI147499, AI143326, AI138764, AI124911 and AI110902 from the National Institutes of Allergy and Infectious Diseases (NIAID) to LBM. Conflict of Interest: The University of California Irvine has filed a patent application on the results reported in this manuscript. No other conflicts exist. Ethical Approval: All subjects were enrolled at the University of California Irvine under Institutional Review Board-approved protocols (IRB # 2020-5779). A written informed consent was received from all participants prior to inclusion in the study.

Persistent pathogens, such as herpes simplex virus 1 (HSV-1), have evolved a variety of immune evasion strategies to avoid being detected and destroyed by the host's immune system. A dynamic cross talk appears to occur between the HSV-1 latency-associated transcript (LAT), the only viral gene that is abundantly transcribed during latency, and the CD8 + T cells that reside in HSV-1 latently infected human and rabbit trigeminal ganglia (TG). The reactivation phenotype of TG that are latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT + TG) is significantly higher than TG latently infected with LAT-null mutant (i.e., LAT − TG). Whether LAT promotes virus reactivation by selectively shaping a unique repertoire of HSV-specific CD8 + T cells in LAT + TG is unknown. In the present study, we assessed the frequency, function, and exhaustion status of TG-resident CD8 + T cells specific to 40 epitopes derived from HSV-1 gB, gD, VP11/12, and VP13/14 proteins, in human leukocyte antigen (HLA-A*0201) transgenic rabbits infected ocularly with LAT + versus LAT – virus. Compared to CD8 + T cells from LAT – TG, CD8 + T cells from LAT + TG (i) recognized a broader selection of nonoverlapping HSV-1 epitopes, (ii) expressed higher levels of PD-1, TIM-3, and CTLA-4 markers of exhaustion, and (iii) produced less tumor necrosis factor alpha, gamma interferon, and granzyme B. These results suggest a novel immune evasion mechanism by which the HSV-1 LAT may contribute to the shaping of a broader repertoire of exhausted HSV-specific CD8 + T cells in latently infected TG, thus allowing for increased viral reactivation. IMPORTANCE A significantly larger repertoire of dysfunctional (exhausted) HSV-specific CD8 + T cells were found in the TG of HLA transgenic rabbits latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT + TG) than in a more restricted repertoire of functional HSV-specific CD8 + T cells in the TG of HLA transgenic rabbits latently infected with LAT-null mutant (i.e., LAT – TG). These findings suggest that the HSV-1 LAT locus interferes with the host cellular immune response by shaping a broader repertoire of exhausted HSV-specific CD8 + T cells within the latency/reactivation TG site.

Emblica officinalis Gaetrn (i.e., Phyllanthus emblica/ Indian gooseberry/ Amla) (EO) has been used extensively as a nutraceutical in several diseases since it is known to boost immunity and offers numerous health benefits such as antioxidant, anti-inflammatory, and anti-aging effects. The goal of our study was to test the hypothesis that EO will rescue human AMD RPE transmitochondrial cells from mitochondria-induced cellular damage. AMD RPE transmitochondrial cell lines were created by fusion of mitochondria DNA-deficient APRE-19 (Rho0) cells with platelets isolated from AMD patients, and therefore had identical nuclei but differed in mitochondrial DNA content. These AMD RPE cells were treated with EO extract followed by characterization of effects of EO using cellular and molecular assays. Herein, EO significantly improved live cell number and mitochondrial membrane potential, reduced apoptosis and oxidative stress, down-regulated VEGF, and up-regulated PGC-1α. In conclusion, EO improved cellular and mitochondrial health, thereby playing a key cytoprotective role in AMD in vitro. Further studies are required to examine the mechanisms that mediate the cytoprotective effects of EO.

Mitochondrial (mt) DNA haplogroups, defined by specific single nucleotide polymorphism (SNPs) patterns, represent populations of diverse geographic origins and may play a role in disparate disease susceptibilities found in different ethnic/racial populations. The most common European haplogroup is H, while the K haplogroup is highly associated with Ashkenazi Jewish populations. Studies using transmitochondrial cybrids (cell lines with identical nuclei but mitochondria from either H or K haplogroup subjects) demonstrated significant molecular and biological differences but mechanisms for these disparities are unclear. In this study, we hypothesized that there is differential retrograde signaling occurring between the Stimulator of Interferon Genes (STING) pathway and H versus K mtDNA haplogroups. Results showed that K cybrids exhibit increased levels of cytoplasmic mtDNA fragments. After STING Knock-Down, H cybrids had lower expression levels for EGFR, BRCA1, DNMT3A, DNMT3B, HDAC1, and IFNα genes, but upregulated DNMT3A compared to control H cybrids. The STING-KD K cybrids showed downregulation of EGFR, DNMT3A, HDAC1, HCAD9, CFH, and CHI, along with upregulation of DNMT1 and IL-6 compared to control K cybrids. Since all cybrids have identical nuclei, the STING DNA sensor system interacts differently with K haplogroup mtDNA compared to H mtDNA for genes related to cancer (EGFR, BRCA1), methylation (DNMT1, DNMT3A, DNMT3B), acetylation (HDAC1, HDCA9), complement (CFH, CHI) and inflammation (IFNα, IL-6). In summary, in non-pathologic conditions, (a) STING is an important retrograde signaling mechanism(s) and (b) cybrids possessing Ashkenazi Jewish mtDNA (K haplogroup) interact with the STING complex differently compared to H cybrids which affects various disease-related pathways.

There is an urgent need for chemical-free and biological-free safe adjuvants to enhance the immunogenicity of vaccines against widespread viral pathogens, such as herpes simplex virus 2 (HSV-2), that infect a large proportion of the world human population. In the present study, we investigated the safety, immunogenicity, and protective efficacy of a laser adjuvant-assisted peptide (LAP) vaccine in the B6 mouse model of genital herpes. This LAP vaccine and its laser-free peptide (LFP) vaccine analog contain the immunodominant HSV-2 glycoprotein B CD8 + T cell epitope (HSV-gB 498–505 ) covalently linked with the promiscuous glycoprotein D CD4 + T helper cell epitope (HSV-gD 49–89 ). Prior to intradermal delivery of the LAP vaccine, the lower-flank shaved skin of B6 or CD11c/eYFP transgenic mice received a topical skin treatment with 5% imiquimod cream and then was exposed for 60 s to a laser, using the FDA-approved nonablative diode. Compared to the LFP vaccine, the LAP vaccine (i) triggered mobilization of dendritic cells (DCs) in the skin, which formed small spots along the laser-treated areas, (ii) induced phenotypic and functional maturation of DCs, (iii) stimulated long-lasting HSV-specific effector memory CD8 + T cells (T EM cells) and tissue-resident CD8 + T cells (T RM cells) locally in the vaginal mucocutaneous tissues (VM), and (iv) induced protective immunity against genital herpes infection and disease. As an alternative to currently used conventional adjuvants, the chemical- and biological-free laser adjuvant offers a well-tolerated, simple-to-produce method to enhance mass vaccination for widespread viral infections. IMPORTANCE Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect a large proportion of the world population. There is an urgent need for chemical-free and biological-free safe adjuvants that would advance mass vaccination against the widespread herpes infections. The present study demonstrates that immunization with a laser-assisted herpes peptide vaccine triggered skin mobilization of dendritic cells (DCs) that stimulated strong and long-lasting HSV-specific effector memory CD8 + T cells (T EM cells) and tissue-resident CD8 + T cells (T RM cells) locally in the vaginal mucocutaneous tissues. The induced local CD8 + T cell response was associated with protection against genital herpes infection and disease. These results draw attention to chemical- and biological-free laser adjuvants as alternatives to currently used conventional adjuvants to enhance mass vaccination for widespread viral infections, such as those caused by HSV-1 and HSV-2.

Most blinding ocular herpetic disease is due to reactivation of herpes simplex virus 1 (HSV-1) from latency rather than to primary acute infection. No herpes simplex vaccine is currently available for use in humans. In this study, we used the HLA-A*02:01 transgenic (HLA Tg) rabbit model of ocular herpes to assess the efficacy of a therapeutic vaccine based on HSV-1 gD epitopes that are recognized mainly by CD8 + T cells from “naturally” protected HLA-A*02:01-positive, HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease). Three ASYMP CD8 + T-cell epitopes (gD 53–61 , gD 70–78 , and gD 278–286 ) were linked with a promiscuous CD4 + T-cell epitope (gD 287–317 ) to create 3 separate pairs of CD4-CD8 peptides, which were then each covalently coupled to an N ε -palmitoyl-lysine moiety, a Toll-like receptor 2 (TLR-2) ligand. This resulted in the construction of 3 CD4-CD8 lipopeptide vaccines. Latently infected HLA Tg rabbits were immunized with a mixture of these 3 ASYMP lipopeptide vaccines, delivered as eye drops in sterile phosphate-buffered saline (PBS). The ASYMP therapeutic vaccination (i) induced HSV-specific CD8 + T cells that prevent HSV-1 reactivation ex vivo from latently infected explanted trigeminal ganglia (TG), (ii) significantly reduced HSV-1 shedding detected in tears, (iii) boosted the number and function of HSV-1 gD epitope-specific CD8 + T cells in draining lymph nodes (DLN), conjunctiva, and TG, and (iv) was associated with fewer exhausted HSV-1 gD-specific PD-1 + TIM-3 + CD8 + T cells. The results underscore the potential of an ASYMP CD8 + T-cell epitope-based therapeutic vaccine strategy against recurrent ocular herpes. IMPORTANCE Seventy percent to 90% of adults harbor herpes simplex virus 1 (HSV-1), which establishes lifelong latency in sensory neurons of the trigeminal ganglia. This latent state sporadically switches to spontaneous reactivation, resulting in viral shedding in tears. Most blinding herpetic disease in humans is due to reactivation of HSV-1 from latency rather than to primary acute infection. To date, there is no licensed therapeutic vaccine that can effectively stop or reduce HSV-1 reactivation from latently infected sensory ganglia and the subsequent shedding in tears. In the present study, we demonstrated that topical ocular therapeutic vaccination of latently infected HLA transgenic rabbits with a lipopeptide vaccine that contains exclusively human “asymptomatic” CD8 + T-cell epitopes successfully decreased spontaneous HSV-1 reactivation, as judged by a significant reduction in spontaneous shedding in tears. The findings should guide the clinical development of a safe and effective T-cell-based therapeutic herpes vaccine.

There is an urgent need for chemical-free and biological-free safe adjuvants to enhance the immunogenicity of vaccines against widespread viral pathogens, such as herpes simplex virus 2 (HSV-2), that infect a large proportion of the world human population. In the present study, we investigated the safety, immunogenicity, and protective efficacy of a laser adjuvant-assisted peptide (LAP) vaccine in the B6 mouse model of genital herpes. This LAP vaccine and its laser-free peptide (LFP) vaccine analog contain the immunodominant HSV-2 glycoprotein B CD8+ T cell epitope (HSV-gB498–505) covalently linked with the promiscuous glycoprotein D CD4+ T helper cell epitope (HSV-gD49–89). Prior to intradermal delivery of the LAP vaccine, the lower-flank shaved skin of B6 or CD11c/eYFP transgenic mice received a topical skin treatment with 5% imiquimod cream and then was exposed for 60 s to a laser, using the FDA-approved nonablative diode. Compared to the LFP vaccine, the LAP vaccine (i) triggered mobilization of dendritic cells (DCs) in the skin, which formed small spots along the laser-treated areas, (ii) induced phenotypic and functional maturation of DCs, (iii) stimulated long-lasting HSV-specific effector memory CD8+ T cells (TEM cells) and tissue-resident CD8+ T cells (TRM cells) locally in the vaginal mucocutaneous tissues (VM), and (iv) induced protective immunity against genital herpes infection and disease. As an alternative to currently used conventional adjuvants, the chemical- and biological-free laser adjuvant offers a well-tolerated, simple-to-produce method to enhance mass vaccination for widespread viral infections.

Mitochondrial damage and epigenetic modifications have been implicated in the pathogenesis of Age-related Macular Degeneration (AMD). This study was designed to investigate the effects of AMD/normal mitochondria on epigenetic regulation in human transmitochondrial retinal pigment epithelial (RPE) cells in vitro. Human RPE cybrid cell lines were created by fusing mitochondria-deficient (Rho0) ARPE-19 cells with platelets obtained from either AMD patients (AMD cybrids) or normal subjects (normal cybrids). Therefore, all cybrids had identical nuclei (derived from ARPE-19 cells) but mitochondria derived from either AMD patients or age-matched normal subjects. AMD cybrids demonstrated increased RNA/protein levels for five methylation-related and four acetylation-related genes, along with lower levels of two methylation and three acetylation genes compared to normal cybrids. Demethylation using 5-Aza-2′-deoxycytidine (DAC) led to decreased expression of VEGF-A gene in AMD cells. Trichostatin A (TSA), an HDAC inhibitor, also influenced protein levels of VEGF-A, HIF1α, NFκB, and CFH in AMD cells. Our findings suggest that retrograde signaling leads to mitochondria-nucleus interactions that influence the epigenetic status of the RPE cells and this may help in the identification of future potential therapeutic targets for AMD.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

Herpes simplex virus 1 (HSV-1) infection is widespread among humans. The HSV-1 virion protein 13/14 (VP13/14), also known as UL47, is a tegument antigen targeted by CD8 + T cells from HSV-seropositive individuals. However, whether VP13/14-specific CD8 + T cells play a role in the natural protection seen in asymptomatic (ASYMP) individuals (individuals who have never had a clinical herpetic disease) has not been elucidated. Using predictive computer-assisted algorithms, we identified 10 potential HLA-A*02:01-restricted CD8 + T-cell epitopes from the 693-amino-acid sequence of the VP13/14 protein. Three out of 10 epitopes exhibited a high to moderate affinity of binding to soluble HLA-A*02:01 molecules. The phenotype and function of CD8 + T cells specific for each epitope were compared in HLA-A*02:01-positive ASYMP individuals and symptomatic (SYMP) individuals (individuals who have frequent clinical herpetic diseases) using determination of a combination of tetramer frequency and the levels of granzyme B, granzyme K, perforin, gamma interferon, tumor necrosis factor alpha, and interleukin-2 production and CD107 a/b cytotoxic degranulation. High frequencies of multifunctional CD8 + T cells directed against three epitopes, VP13/14 from amino acids 286 to 294 (VP13/14 286–294 ), VP13/14 from amino acids 504 to 512 (VP13/14 504–512 ), and VP13/14 from amino acids 544 to 552 (VP13/14 544–552 ), were detected in ASYMP individuals, while only low frequencies were detected in SYMP individuals. The three epitopes also predominantly recalled more CD45RA low CD44 high CCR7 low CD62L low CD8 + effector memory T cells (T EM cells) in ASYMP individuals than SYMP individuals. Moreover, immunization of HLA-A*02:01 transgenic mice with the three CD8 + T EM -cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8 + T EM cells associated with strong protective immunity against ocular herpesvirus infection and disease. Our findings outline the phenotypic and functional features of protective HSV-specific CD8 + T cells that should guide the development of a safe and effective T-cell-based herpes simplex vaccine. IMPORTANCE Although most herpes simplex virus 1 (HSV-1)-infected individuals shed the virus in their body fluids following reactivation from latently infected sensory ganglia, the majority never develop a recurrent herpetic disease and remain asymptomatic (ASYMP). In contrast, small proportions of individuals are symptomatic (SYMP) and develop frequent bouts of recurrent disease. The present study demonstrates that naturally protected ASYMP individuals have a higher frequency of effector memory CD8 + T cells (CD8 + T EM cells) specific to three epitopes derived from the HSV-1 tegument protein VP13/14 (VP13/14 286–294 ,VP13/14 504–512 , and VP13/14 544–552 ) than SYMP patients. Moreover, immunization of humanized HLA-A*02:01 transgenic mice with the three CD8 + T EM -cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8 + T cells associated with strong protective immunity against ocular herpesvirus infection and disease. The findings support the emerging concept of the development of a safe and effective asymptomatic herpes simplex vaccine that is selectively based on CD8 + T-cell epitopes from ASYMP individuals.

Professor Steven L. Wechsler, a world-renowned eye researcher and virologist, passed away unexpectedly on June 12, 2016 at the age of 68. Many scientists came to know Professor Wechsler as a gifted researcher in the field of ocular Herpes Simplex Virus (HSV-1) latency, reactivation, and pathogenesis. Professor Wechsler published over 150 peer-reviewed scientific papers during his career, pushing forward the frontiers of his field eye research. His colleagues would say, 'Steve literally wrote the book on herpes latency and reactivation.' He was the first to show that the HSV-1 latency-associated transcript (LAT) is essential for the HSV-1 high spontaneous reactivation phenotype and that LAT has anti-apoptosis activity. This discovery of LAT's anti-apoptosis activity, which is a key factor in how the LAT gene enhances reactivation, was published in Science in 2000 and created a new paradigm that greatly increased understanding of HSV-1 latency and reactivation. In collaboration with Professor Lbachir BenMohamed, an immunologist, they later demonstrated that LAT also acts as an immune evasion gene. He was a caring scientist who truly enjoyed working and sharing his experience and expertise with young researchers. He will be remembered as a significant pillar within scientific and ocular herpes research communities worldwide. Professor Wechsler's dedication to science, his compassionate character, and wonderful sense of humor were exemplary. We, who were his friends and colleagues, will mourn his passing deeply.

Herpes simplex virus 1 (HSV-1) infection is widespread among humans. The HSV-1 virion protein 13/14 (VP13/14), also known as UL47, is a tegument antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether VP13/14-specific CD8+ T cells play a role in the natural protection seen in asymptomatic (ASYMP) individuals (individuals who have never had a clinical herpetic disease) has not been elucidated. Using predictive computer-assisted algorithms, we identified 10 potential HLA-A*02:01-restricted CD8+ T-cell epitopes from the 693-amino-acid sequence of the VP13/14 protein. Three out of 10 epitopes exhibited a high to moderate affinity of binding to soluble HLA-A*02:01 molecules. The phenotype and function of CD8+ T cells specific for each epitope were compared in HLA-A*02:01-positive ASYMP individuals and symptomatic (SYMP) individuals (individuals who have frequent clinical herpetic diseases) using determination of a combination of tetramer frequency and the levels of granzyme B, granzyme K, perforin, gamma interferon, tumor necrosis factor alpha, and interleukin-2 production and CD107a/b cytotoxic degranulation. High frequencies of multifunctional CD8+ T cells directed against three epitopes, VP13/14 from amino acids 286 to 294 (VP13/14286–294), VP13/14 from amino acids 504 to 512 (VP13/14504–512), and VP13/14 from amino acids 544 to 552 (VP13/14544–552), were detected in ASYMP individuals, while only low frequencies were detected in SYMP individuals. The three epitopes also predominantly recalled more CD45RAlow CD44high CCR7low CD62Llow CD8+ effector memory T cells (TEM cells) in ASYMP individuals than SYMP individuals. Moreover, immunization of HLA-A*02:01 transgenic mice with the three CD8+ TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8+ TEM cells associated with strong protective immunity against ocular herpesvirus infection and disease. Our findings outline the phenotypic and functional features of protective HSV-specific CD8+ T cells that should guide the development of a safe and effective T-cell-based herpes simplex vaccine.

Following ocular herpes simplex virus 1 (HSV-1) infection of C57BL/6 mice, HSV-specific (HSV-gB 498–505 tetramer + ) CD8 + T cells are induced, selectively retained in latently infected trigeminal ganglia (TG), and appear to decrease HSV-1 reactivation. The HSV-1 latency-associated transcript (LAT) gene, the only viral gene that is abundantly transcribed during latency, increases reactivation. Previously we found that during latency with HSV-1 strain McKrae-derived viruses, more of the total TG resident CD8 T cells expressed markers of exhaustion with LAT + virus compared to LAT − virus. Here we extend these findings to HSV-1 strain 17syn+-derived LAT + and LAT − viruses and to a virus expressing just the first 20% of LAT. Thus, the previous findings were not an artifact of HSV-1 strain McKrae, and the LAT function involved mapped to the first 1.5 kb of LAT. Importantly, to our knowledge, we show here for the first time that during LAT + virus latency, most of the HSV-1-specific TG resident CD8 T cells were functionally exhausted, as judged by low cytotoxic function and decreased gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. This resulted in LAT − TG having more functional HSV-gB 498–505 tetramer + CD8 + T cells compared to LAT + TG. In addition, LAT expression, in the absence of other HSV-1 gene products, appeared to be able to directly or indirectly upregulate both PD-L1 and major histocompatibility complex class I (MHC-I) on mouse neuroblastoma cells (Neuro2A). These findings may constitute a novel immune evasion mechanism whereby the HSV-1 LAT directly or indirectly promotes functional exhaustion (i.e., dysfunction) of HSV-specific CD8 + T cells in latently infected TG, resulting in increased virus reactivation.

Both topical ocular and topical intranasal immunizations have been reported to stimulate the ocular mucosal immune system (OMIS) and the systemic immune system. Nasolacrimal ducts (NLDs) are the connecting bridges between the OMIS and nasal cavity-associated lymphoid tissue (NALT). These ducts drain topical ocularly administrated solutions into the inferior meatus of the nose to reach the NALT. Inversely, NLDs also drain intranasally administrated solutions to the mucosal surface of the eye and thus the OMIS. This unique anatomical connection between the OMIS and NALT systems provoked us to test whether the OMIS and NALT are immunologically interdependent. In this report, we show that both topical ocular administration and topical intranasal administration of a mixture of immunodominant CD4+T-cell epitope peptides from herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) emulsified with the CpG2007mucosal adjuvant are capable of inducing local (in conjunctiva) as well as systemic (in spleen) HSV-peptide-specific CD4+T-cell responses. Interestingly, surgical closure of NLDs did not significantly alter local ocular mucosal CD4+T-cell responses induced following topical ocular immunization but did significantly enhance systemic CD4+T-cell responses (as measured by both T-cell proliferation and gamma interferon (IFN-γ) production;P< 0.005). In contrast, NLD closure significantly decreased ocular mucosal, but not systemic, CD4+T-cell responses following intranasal administration of the same vaccine solution (P< 0.001). The study suggests that NALT and the OMIS are immunologically interconnected.

Breast cancer (BrCa) is the second leading cause of cancer-related deaths for women worldwide. Evidence from both patients and mouse cancer models suggests that the simultaneous induction of BrCa-specific CD4(+) T cells, CD8(+) cytotoxic T cells, and antibodies is crucial for providing immune resistance. However, almost all current vaccines address only a single arm of the immune system, which may explain their lack of efficacy. We believe that the correct response to monovalent vaccines' "failure" is to increase our knowledge about antitumor protective immunity and to develop a multivalent vaccine molecule that can simultaneously induce multiple arms of the immune system. We highlight here recent advances in anti-BrCa peptide-based vaccine strategies with an emphasis on the self adjuvanting multivalent glycolipopeptide vaccine strategy recently developed in our laboratory and which showed promising results in both immunotherapeutic and immunoprophylactic settings.

Evidence obtained from both animal models and humans suggests that T cells specific for HSV-1 and HSV-2 glycoprotein D (gD) contribute to protective immunity against herpes infection. However, knowledge of gD-specific human T cell responses is limited to CD4+ T cell epitopes, with no CD8+ T cell epitopes identified to date. In this study, we screened the HSV-1 gD amino acid sequence for HLA-A*0201-restricted epitopes using several predictive computational algorithms and identified 10 high probability CD8+ T cell epitopes. Synthetic peptides corresponding to four of these epitopes, each nine to 10 amino acids in length, exhibited high-affinity binding in vitro to purified human HLA-A*0201 molecules. Three of these four peptide epitopes, gD53–61, gD70–78, and gD278–286, significantly stabilized HLA-A*0201 molecules on T2 cell lines and are highly conserved among and between HSV-1 and HSV-2 strains. Consistent with this, in 33 sequentially studied HLA-A*0201-positive, HSV-1-seropositive, and/or HSV-2-seropositive healthy individuals, the most frequent and robust CD8+ T cell responses, assessed by IFN-γ ELISPOT, CD107a/b cytotoxic degranulation, and tetramer assays, were directed mainly against gD53–61, gD70–78, and gD278–286 epitopes. In addition, CD8+ T cell lines generated by gD53–61, gD70–78, and gD278–286 peptides recognized infected target cells expressing native gD. Lastly, CD8+ T cell responses specific to gD53–61, gD70–78, and gD278–286 epitopes were induced in HLA-A*0201 transgenic mice following ocular or genital infection with either HSV-1 or HSV-2. The functional gD CD8+ T cell epitopes described herein are potentially important components of clinical immunotherapeutic and immunoprophylactic herpes vaccines.

Purpose Variations in mitochondrial DNA (mtDNA) and abnormalities in the complement pathways have been implicated in the pathogenesis of age-related macular degeneration (AMD). This study was designed to determine the effects of mtDNA from AMD subjects on the complement pathway. Methods Transmitochondrial cybrids were prepared by fusing platelets from AMD and age-matched Normal subjects with Rho0 (lacking mtDNA) human ARPE-19 cells. Quantitative PCR and Western blotting were performed to examine gene and protein expression profiles, respectively, of complement markers in these cybrids. Bioenergetic profiles of Normal and AMD cybrids were examined using the Seahorse XF24 flux analyzer. Results Significant decreases in the gene and protein expression of complement inhibitors, along with significantly higher levels of complement activators, were found in AMD cybrids compared to Older-Normal cybrids. Seahorse flux data demonstrated that the bioenergetic profiles for Older-Normal and Older-AMD cybrid samples were similar to each other but were lower compared to Young-Normal cybrid samples. Conclusion In summary, since all cybrids had identical nuclei and differed only in mtDNA content, the observed changes in components of complement pathways can be attributed to mtDNA variations in the AMD subjects, suggesting that mitochondrial genome and retrograde signaling play critical roles in this disease. Furthermore, the similar bioenergetic profiles of AMD and Older-Normal cybrids indicate that the signaling between mitochondria and nuclei are probably not via a respiratory pathway.

We studied the phenotype and distribution of “naturally” occurring CD4+CD25+T regulatory cells (CD4+CD25+nTregcells) resident in rabbit conjunctiva, the main T-cell inductive site of the ocular mucosal immune system, and we investigated their suppressive capacities using herpes simplex virus type 1 (HSV-1)-specific effector T (Teff) cells induced during ocular infection. The expression of CD4, CD25, CTLA4, GITR, and Foxp3 was examined by reverse transcription-PCR, Western blotting, and fluorescence-activated cell sorter analysis in CD45+pan-leukocytes isolated from conjunctiva, spleen, and peripheral blood monocyte cells (PBMC) of HSV-1-infected and uninfected rabbits. Normal conjunctiva showed a higher frequency of CD4+CD25(Bright+)T cells than did spleen and PBMC. These cells expressed high levels of Foxp3, GITR, and CTLA4 molecules. CD4+CD25(Bright+)T cells were localized continuously along the upper and lower palpebral and bulbar conjunctiva, throughout the epithelium and substantia propria. Conjunctiva-derived CD4+CD25(Bright+)T cells, but not CD4+CD25(low)T cells, efficiently suppressed HSV-specific CD4+and CD8+Teffcells. The CD4+CD25(Bright+)T-cell-mediated suppression was effective on both peripheral blood and conjunctiva infiltrating Teffcells and was cell-cell contact dependent but independent of interleukin-10 and transforming growth factor β. Interestingly, during an ocular herpes infection, there was a selective increase in the frequency and suppressive capacity of Foxp3+CD4+CD25(Bright+)T cells in conjunctiva but not in the spleen or in peripheral blood. Altogether, these results provide the first evidence that functional Foxp3+CD4+CD25(Bright+)Tregcells accumulate in the conjunctiva. It remains to be determined whether conjunctiva CD4+CD25+nTregcells affect the topical/mucosal delivery of subunit vaccines that stimulate the ocular mucosal immune system.

Purpose To determine the association between complement factor H (CFH) polymorphism T1277C (tyrosine-402 → histidine-402) and phenotypic variations of age-related macular degeneration (AMD). Design Cross-sectional observational study. Participants Subjects with dry or wet AMD and a control population consisting of age-matched non-AMD subjects from 2 clinical facilities examined during the period January 1, 1999 through December 31, 2002. Methods Total DNA isolated from the leukocytes of 66 AMD subjects and 58 age-matched control subjects was studied. The CFH gene was amplified by polymerase chain reaction and analyzed by Nla III restriction fragment length analysis. Main Outcome Measures Incidence of CHF polymorphism with the occurrence of AMD. Results Among the AMD patients, 15 had dry and 51 had wet AMD. For the CFH gene, the T1277C variant showed the genotype distribution as CC, TC, and TT. There was a strong association between homozygous C and AMD compared with the control population (odds ratio [OR] = 3.4; 95% confidence interval [CI], 1.32–8.74; P = 0.0053). Furthermore, dry AMD had a stronger association (OR, 8.32; 95% CI, 2.30–30.11; P = 0.001) than wet AMD (OR, 2.49; 95% CI, 0.90–6.84; P = 0.039) compared with the control population. Homozygous T was more prevalent in the control subjects compared with AMD patients (OR, 5; 95% CI, 2.18–11.43; P = 0.00005). Conclusions Complement factor H polymorphism T1277C (tyrosine-402 → histidine-402) is strongly associated with both dry and wet AMD and points to a possible role for inflammation in the pathogenesis of AMD.

Immunology and Microbiology A Herpes Simplex Virus Type 1 Human Asymptomatic CD8 þ T-Cell Epitopes-Based Vaccine Protects Against Ocular Herpes in a ‘‘Humanized’’ HLA Transgenic Rabbit Model Ruchi Srivastava, 1 Arif A. Khan, 1 Jiawei Huang, 1 Anthony B. Nesburn, 1 Steven L. Wechsler, 1–3 and Lbachir BenMohamed 1,4,5 1 Laboratory of Cellular and Molecular Immunology, Gavin Herbert Eye Institute, University of California Irvine, School of Medicine, Irvine, California, United States Department of Microbiology and Molecular Genetics, University of California Irvine, School of Medicine, Irvine, California, United States 3 Center for Virus Research, University of California Irvine, Irvine, California, United States Department of Molecular Biology & Biochemistry Institute for Immunology, University of California Irvine, School of Medicine, Irvine, California, United States Correspondence: Lbachir BenMo- hamed, Laboratory of Cellular and Molecular Immunology, Gavin Her- bert Eye Institute; Ophthalmology Research Laboratories, Hewitt Hall, Room 2032, 843 Health Sciences Road, Irvine, CA 92697-4390, USA; Lbenmoha@uci.edu. RS and AAK contributed equally to the work presented here and should therefore be regarded as equivalent authors. Submitted: April 10, 2015 Accepted: May 23, 2015 Citation: Srivastava R, Khan AA, Huang J, Nesburn AB, Wechsler SL, BenMohamed L. A herpes simplex virus type 1 human asymptomatic CD8 þ T-cell epitopes-based vaccine protects against ocular herpes in a ‘‘humanized’’ HLA transgenic rabbit model. Invest Ophthalmol Vis Sci. 2015;56:4013–4028. DOI:10.1167/ iovs.15-17074 P URPOSE . A clinical vaccine that protects from ocular herpes simplex virus type 1 (HSV-1) infection and disease still is lacking. In the present study, preclinical vaccine trials of nine asymptomatic (ASYMP) peptides, selected from HSV-1 glycoproteins B (gB), and tegument proteins VP11/12 and VP13/14, were performed in the ‘‘humanized’’ HLA–transgenic rabbit (HLA-Tg rabbit) model of ocular herpes. We recently reported that these peptides are highly recognized by CD8 þ T cells from ‘‘naturally’’ protected HSV-1–seropositive healthy ASYMP individuals (who have never had clinical herpes disease). M ETHODS . Mixtures of three ASYMP CD8 þ T-cell peptides derived from either HSV-1 gB, VP11/ 12, or VP13/14 were delivered subcutaneously to different groups of HLA-Tg rabbits (n ¼ 10) in incomplete Freund’s adjuvant, twice at 15-day intervals. The frequency and function of HSV-1 epitope-specific CD8 þ T cells induced by these peptides and their protective efficacy, in terms of survival, virus replication in the eye, and ocular herpetic disease were assessed after an ocular challenge with HSV-1 (strain McKrae). R ESULTS . All mixtures elicited strong and polyfunctional IFN-c– and TNF-a–producing CD107 þ CD8 þ cytotoxic T cells, associated with a significant reduction in death, ocular herpes infection, and disease (P < 0.015). C ONCLUSIONS . The results of this preclinical trial support the screening strategy used to select the HSV-1 ASYMP CD8 þ T-cell epitopes, emphasize their valuable immunogenic and protective efficacy against ocular herpes, and provide a prototype vaccine formulation that may be highly efficacious for preventing ocular herpes in humans. Keywords: HSV-1, HLA transgenic rabbit, animal model, T cell, glycoprotein B, VP11/12, VP13/14, vaccine, ocular ith a staggering one billion individuals worldwide currently carrying herpes simplex virus type 1 (HSV-1), herpes remains one of the most prevalent viral infections of the eye. 1–7 Ocular herpes infection causes a spectrum of clinical manifestations ranging from blepharitis, conjunctivitis, and dendritic keratitis to disciform stromal edema and blinding stromal keratitis (HSK). 8,9 In the United States alone, over 450,000 people have a history of recurrent ocular HSV requiring doctor visits, antiviral drug treatments, and, in severe cases, corneal transplants. 10–12 Despite the availability of many intervention strategies, the global picture for ocular herpes continues to deteriorate. 13 Current antiviral drug therapies (e.g., acyclovir and derivatives) do not eliminate the virus and reduce recurrent herpetic disease by only approximately 45%. 14 The development of an effective vaccine would present an unparalleled alternative to antiviral drugs, as it would be a powerful and cost-effective means to reduce HSV-1 ocular infection and lessen associated blinding ocular herpetic disease (reviewed previously 1 ). Our long-term goal is to develop a vaccine to prevent HSV-1 infection and protect against ocular herpes disease. The most recent vaccine clinical trials that used recombinant HSV proteins failed to protect despite inducing strong HSV-specific neutralizing antibodies. These failures emphasize three major gaps in knowledge: the need to induce strong T-cell responses (in addition to humoral responses) for protection against ocular herpes, 15 the need to identify protective human herpes T-cell epitopes from HSV antigens (Ags) to be incorporated in the next generation HSV vaccines, 15 and the preclinical evaluation of protective efficacy of human herpes T-cell epitopes in a reliable animal model of ocular herpes infection and disease. One ‘‘common denominator’’ among previously failed vaccine clinical trials is that they either used the whole virus or whole HSV proteins (e.g., HSV glycoprotein B and/or D [gB Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc. iovs.arvojournals.org j ISSN: 1552-5783 W

Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and -J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2'-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases.

Molecularly defined vaccine formulations capable of inducing antiviral CD8 + T-cell-specific immunity in a manner compatible with human delivery are limited. Few molecules achieve this target without the support of an appropriate immunological adjuvant. In this study, we investigate the potential of totally synthetic palmitoyl-tailed helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL chimeric lipopeptides) to induce herpes simplex virus type 1 (HSV-1)-specific CD8 + T-cell responses. As a model antigen, the HSV-1 glycoprotein B (498-505) (gB 498-505 ) CD8 + CTL epitope was synthesized in line with the Pan DR peptide (PADRE), a universal CD4 + Th epitope. The peptide backbone, composed solely of both epitopes, was extended by N-terminal attachment of one (PAM-Th-CTL), two [(PAM) 2 -Th-CTL], or three [(PAM) 3 -Th-CTL] palmitoyl lysines and delivered to H2 b mice in adjuvant-free saline. Potent HSV-1 gB 498-505 -specific antiviral CD8 + T-cell effector type 1 responses were induced by each of the palmitoyl-tailed Th-CTL chimeric epitopes, irrespective of the number of lipid moieties. The palmitoyl-tailed Th-CTL chimeric epitopes provoked cell surface expression of major histocompatibility complex and costimulatory molecules and production of interleukin-12 and tumor necrosis factor alpha proinflammatory cytokines by immature dendritic cells. Following ocular HSV-1 challenge, palmitoyl-tailed Th-CTL-immunized mice exhibited a decrease of virus replication in the eye and in the local trigeminal ganglion and reduced herpetic blepharitis and corneal scarring. The rational of the molecularly defined vaccine approach presented in this study may be applied to ocular herpes and other viral infections in humans, providing steps are taken to include appropriate Th and CTL epitopes and lipid groups.

LAT (latency-associated transcript) is the only herpes simplex virus type 1 (HSV-1) transcript abundantly expressed during neuronal latency. LAT expression is required for the high reactivation phenotype of HSV-1 and this phenotype correlates with LAT's anti-apoptosis properties. LAT nucleotides 1 to 1499 inhibit caspase-8 (death receptor apoptotic pathway), but not caspase-9 (mitochondrial apoptotic pathway), -induced apoptosis as efficiently as larger LAT fragments. LAT sequences important for inhibiting caspase-8-induced apoptosis were also localized. The ability of LAT nucleotides 1 to 1499 to efficiently inhibit caspase-8-induced apoptosis correlates with the high reactivation phenotype of a mutant virus expressing just the first 1.5 kb of LAT (nucleotides 1 to 1499).

The herpes simplex virus type 1 latency-associated transcript (LAT) inhibits apoptosis. We demonstrate here that LAT influences the accumulation of the Bcl-x L transcript versus the Bcl-x S transcript in Neuro-2A cells. Bcl-x L encodes an antiapoptotic protein, whereas Bcl-x S encodes a proapoptotic protein. Promoting the accumulation of Bcl-x L in neurons may inhibit apoptosis, thus enhancing the latency-reactivation cycle.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for the high spontaneous and induced reactivation phenotype of HSV-1 in the rabbit ocular model and for the high induced reactivation phenotype in the mouse ocular model. Recently we showed that LAT has an antiapoptosis function, and we hypothesized that LAT's ability to inhibit apoptosis played an important role in LAT's ability to enhance the reactivation phenotype. Expression of just the first 1.5 kb of the 8.3-kb LAT gene is sufficient for both inhibition of apoptosis in an in vitro transient-transfection assay and the high spontaneous reactivation phenotype in vivo. Here we show the results of more complex mapping studies in which inhibition of apoptosis and the enhanced spontaneous reactivation phenotype also appear to be linked. The HSV-1 mutant virus dLAT371 has a high spontaneous reactivation phenotype in rabbits, suggesting that the LAT region deleted in this mutant (LAT nucleotides 76 to 447) is not required for this phenotype. The LAT3.3A viral mutant (which expresses LAT nucleotides 1 to 1499) also has a high spontaneous reactivation phenotype, suggesting that the region of LAT not expressed by this mutant (LAT nucleotide 1500 to the end of LAT) is also not required for this phenotype. Surprisingly, LAT2.9A, which is a combination of dLAT371 and LAT3.3A (i.e., it expresses LAT nucleotides 1 to 76 and 447 to 1499), has a low spontaneous reactivation phenotype indistinguishable from that of LAT null mutants. We report here that consistent with the low spontaneous reactivation phenotype of LAT2.9A, a plasmid expressing the identical LAT RNA did not inhibit caspase 9-induced apoptosis. In contrast, plasmids containing the same deletion but able to transcribe up to or past LAT nucleotide 2850 (rather than just up to LAT nucleotide 1499) inhibited caspase 9-induced apoptosis, consistent with the high spontaneous reactivation phenotype of dLAT371. Thus, LAT2.9A may have a low spontaneous reactivation phenotype because the LAT RNA that is made cannot block apoptosis, and dLAT371 apparently has a high spontaneous reactivation phenotype because the LAT RNA made has significant antiapoptosis activity. Furthermore, LAT appeared to have at least two regions capable of interfering with caspase 9-induced apoptosis. One region partially overlaps LAT nucleotides 76 to 447. The second region is partially (or completely) downstream of LAT nucleotide 1499.

Studies have shown an intimate relationship between cholesterol and retinal diseases; we examined the effects of cholesterol oxides on cultured cells. Using the rat retinal precursor cell line R28 and the human RPE cell line ARPE-19, we investigated the potential cytotoxicity of cholesterol oxides. Cultured R28 and ARPE-19 cells were treated with either 25-hydroxycholesterol and 7-ketocholesterol (0-50 microg/ml). Cell viability was determined by the WST-1 colorimetric assay. Production of reactive oxygen intermediate (ROI) was assessed by a fluorescent probe-based assay (2',7'-dichlorodihydrofluorescein diacetate [H2DCFDA]). To detect the presence of apoptosis, DNA fragmentation gel analysis and Hoescht nuclear staining were performed. Both cholesterol oxides tested were toxic in a time- and dose-dependent fashion to the two cell lines used in this study. Treatment of R28 cells with either 25-hydroxycholesterol or 7-ketocholesterol at a concentration of 25 micro/ml resulted in greater than 50% loss of cell viability after 24 h. ARPE-19 cells were slightly less affected, with a loss of cell viability of approximately 20% and 40% after 24 h-exposure of 25-hydroxycholesterol and 7-ketocholesterol, respectively. DNA fragmentation and chromatin condensation demonstrated apoptotic events occurring in 7-ketocholesterol-treated cells. The fluorescent assay for ROI production showed that after an hour of exposure to 7-ketocholesterol, R28 cells responded with increased levels of ROIs, whereas no immediate production of ROIs were detected with treated ARPE-19 cells. These in vitro findings provide evidence that cholesterol oxides can directly damage cultured retinal and RPE cells. The oxysterol-induced oxidative stress in these cells may be a factor in the pathology of retinal degenerative diseases.

The HSV type 1 tegument virion phosphoprotein (VP) 11/12 (VP11/12) is a major Ag targeted by CD8+ T cells from HSV-seropositive individuals. However, whether and which VP11/12 epitope–specific CD8+ T cells play a role in the “natural” protection seen in seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01–restricted CD8+ T cell epitopes from the 718-aa sequence of VP11/12. Three of 10 epitopes exhibited high-to-moderate binding affinity to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01–positive and HSV-1–seropositive ASYMP individuals, the most frequent, robust, and polyfunctional effector CD8+ T cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107a/b cytotoxic degranulation, IFN-γ, and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266–74, VP11/12220–228, and VP11/12702–710. Interestingly, ASYMP individuals had a significantly higher proportion of CD45RAlowCCR7lowCD44highCD62LlowCD27lowCD28lowCD8+ effector memory CD8+ T cells (TEMs) specific to the three epitopes, compared with symptomatic individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8+ TEM cell epitopes induced robust and polyfunctional epitope-specific CD8+ TEM cells that were associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSV-specific CD8+ T cells that should guide the development of an effective T cell–based herpes vaccine.

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8 + T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8 + T cells play a key role in the “natural” protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8 + T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found the following. (i) Healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8 + T cells (T EM cells) (CD45RA low CCR7 low CD44 high CD62L low ). In contrast, SYMP patients had frequent less-differentiated central memory CD8 + T cells (T CM cells) (CD45RA low CCR7 high CD44 low CD62L high ). (ii) ASYMP individuals had significantly higher proportions of multifunctional effector CD8 + T cells which responded mainly to gB 342–350 and gB 561–569 “ASYMP” epitopes, and simultaneously produced IFN-γ, CD107 a/b , granzyme B, and perforin. In contrast, effector CD8 + T cells from SYMP individuals were mostly monofunctional and were directed mainly against nonoverlapping gB 17–25 and gB 183–191 “SYMP” epitopes. (iii) Immunization of an HLA-A*02:01 transgenic mouse model of ocular herpes with “ASYMP” CD8 + T EM cell epitopes, but not with “SYMP” CD8 + T CM cell epitopes, induced a strong CD8 + T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8 + T EM cells in protection against herpes and should be considered in the development of an effective vaccine. IMPORTANCE A significantly higher proportion of differentiated and multifunctional HSV-1 gB-specific effector memory CD8 + T cells (T EM cells) (CD45RA low CCR7 low CD44 high CD62L low ) were found in healthy ASYMP individuals who are seropositive for HSV-1 but never had any recurrent herpetic disease, while there were frequent less-differentiated and monofunctional central memory CD8 + T cells (T CM cells) (CD45RA low CCR7 high CD44 low CD62L high ) in SYMP patients. Immunization with “ASYMP” CD8 + T EM cell epitopes, but not with “SYMP” CD8 + T CM cell epitopes, induced a strong protective HSV-specific CD8 + T cell response in HLA-A*02:01 transgenic mice. These findings are important for the development of a safe and effective T cell-based herpes vaccine.

Evidence suggests that in BALB/c mice infected with HSV-1, increased corneal scarring correlated with the presence of IL-12p40 mRNA in the cornea. To determine if this observed correlation reflected function, we have utilized mice with a homologous disruption of the gene encoding either the IL-12p35 subunit or the IL-12p40 subunit of IL-12. The severity of corneal scarring following ocular infection with HSV-1 was reduced significantly in naive IL-12p35- and IL-12p40-deficient mice compared with naive BALB/c mice, with the corneal scarring being low grade in the IL-12p35-deficient mice and completely absent in the IL-12p40-deficient mice. The reduction in the corneal scarring could not be attributed to a reduction in the HSV-1 titers in the eyes, which were not significantly different from the BALB/c mice, or to differences in the production of TH1 responses (IL-2 and IFN-γ production) by the infected mice. Taken together, these results suggest the importance of IL-12 in the induction of corneal scarring in HSV-1-infected mice.

The expression of interleukin-2 (IL-2) has been implicated in the modulation of the outcome of ocular infection with herpes simplex virus type 1 (HSV-1); however, its effects remain controversial. To clarify the role of IL-2, we constructed a recombinant HSV-1 (HSV-IL-2) that expresses two copies of the murine IL-2 gene under the control of the latency-associated transcript (LAT) promoter of HSV-1 in a LAT-negative virus. In tissue culture, the replication of the HSV-IL-2 was 100-fold lower than that of the wild-type virus at a low multiplicity of infection (MOI). Addition of recombinant anti-IL-2 polyclonal antibody markedly enhanced HSV-IL-2 replication in tissue culture. In the 7-day period after ocular infection of BALB/c mice, the replication of HSV-IL-2 was significantly lower than that of wild-type virus in tear cultures, whole eyes, and brain, but was equivalent to wild-type replication in the trigeminal ganglia. Ocular challenge of BALB/c mice with HSV-IL-2 alone, at an MOI that resulted in only 13% survival when parental virus was used, was associated with 90% survival. This decrease in virulence was further shown to be attributable to the expression of IL-2 by coinfection of mice with HSV-IL-2 and the parental virus. This resulted in a decrease in virulence of the parental virus (5% survival when administered alone versus 50% survival on coinfection with HSV-IL-2). The survival of HSV-IL-2-infected mice was compromised by depletion of either IL-2, CD4+, or CD8+T cells (50% survival) and abolished completely by depletion of both T-cell subtypes. Moreover, depletion of CD4+T cells, CD8+T cells, or both increased the titers of HSV-IL-2 in the tears, eyes, trigeminal ganglia, and brains of infected mice, so that titers were equivalent to or higher than that of the parental virus. These results suggest that IL-2 expression by recombinant HSV-1 reduces virulence and that depletion of IL-2 or T cells increases virulence in HSV-1-infected mice.