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Introduction: Since early 2020, several SARS-CoV-2 variants of concern (VOCs) continue to emerge, evading waning antibody mediated immunity produced by the current Spike-alone based COVID-19 vaccines. This caused a prolonged and persistent COVID-19 pandemic that is going to enter its fifth year. Thus, the need remains for innovative next generation vaccines that would incorporate protective Spike-derived B-cell epitopes that resist immune evasion., Methods: Towards that goal, in this study we (i) Screened the sequences of Spike among many VOCs and identified conserved and non-conserved linear B-cell epitopes; (ii) Compared titers and neutralization antibodies specific to these conserved and non-conserved B-cell epitopes from serum of symptomatic and asymptomatic COVID-19 patients that were exposed to multiple VOCs across the 5-year COVID-19 pandemic, and (iii) Compared protective efficacy of conserved versus non-conserved B-cell epitopes against the most pathogenic Delta variant in a "humanized" ACE-2/HLA transgenic mouse model., Results: We found robust conserved B-cell epitope-specific antibody titers and neutralization in sera from asymptomatic COVID-19 patients. In contrast, sera from symptomatic patients contained weaker antibody responses specific to conserved B-cell epitopes. A multi-epitope COVID-19 vaccine that incorporated the conserved B-cell epitopes, but not the non-conserved B-cell epitopes, significantly protected the ACE2/HLA transgenic mice against infection and COVID-19 like symptoms caused by the Delta variant., Discussion: These findings underscore the importance of conserved B-cell epitopes in generating robust protective immunity against severe COVID-19 symptoms caused by various VOCs, providing valuable insights for the development of broad-spectrum next generation Coronavirus vaccines capable of conferring cross-variant protective immunity., Competing Interests: Authors HV, DG, JU and LB were employed by the company TechImmune, LLC. Studies of this report were supported 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 LB and by R43AI174383 to TechImmune, LLC. LB has an equity interest in TechImmune, LLC., a company that may potentially benefit from the research results and serves on the company’s Scientific Advisory Board. LB’s relationship with TechImmune, LLC., has been reviewed and approved by the University of California, Irvine in accordance with its conflict-of-interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2025 Zayou, Prakash, Vahed, Dhanushkodi, Quadiri, Belmouden, Lemkhente, Chentoufi, Gil, Ulmer and BenMohamed.)

Background: Nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) have emerged as a promising vaccine strategy, especially for COVID-19. While the LNPs protect mRNA from degradation and efficiently deliver the mRNA to antigen-presenting cells, the effect of lipid composition on the immunogenicity and protective efficacy of mRNA/LNP vaccines is not well characterized. Studies on using the mRNA/LNP platform for vaccines have largely focused on the nucleic acid cargo with less attention paid to the LNP vehicle. Whether the composition and biophysical properties of LNPs impact vaccine performance remains to be fully elucidated., Methods: In the present study, we used SARS-CoV-2 Spike-mRNA as a prototype vaccine to study the effect of four different LNPs with various lipid compositions., Results: We demonstrate that when the same Spike-mRNA was delivered in the LNP4 formulation based on phospholipid 1,2-dioleoyl-sn-glycero-3-Phosphoethanolamine, it outperformed other LNPs (LNP1, LNP2, and LNP3) that are based on different lipids. Compared to the other three LNPs, LNP4 (i) enhanced the phenotypic and functional maturation of dendritic cells; (ii) induced strong T-cell responses; (iii) increased the secretion of proinflammatory cytokines and pro-follicular T helper (Tfh) cell cytokines; (iv) induced higher neutralization IgG titers; and (v) provided better protection against SARS-CoV-2 infection and COVID-19-like symptoms in the hamster model. Furthermore, we compared LNP-4 with the commercially available LNPs and found it to provide better T-cell immunity against COVID-19 in hamsters., Conclusion: This study suggests mRNA vaccines encapsulated in Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine containing LNPs induced Potent B- and T cell immunity. The mechanisms by which Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine-based LNPs may activate protective B and T cells are discussed.

Since early 2020, several SARS-CoV-2 variants of concern (VOCs) continue to emerge, evading waning antibody mediated immunity produced by the current Spike-alone based COVID-19 vaccines. This caused a prolonged and persistent COVID-19 pandemic that is going to enter its fifth year. Thus, the need remains for innovative next generation vaccines that would incorporate protective Spike-derived B-cell epitopes that resist immune evasion. Towards that goal, in this study we ( i ) Screened the sequences of Spike among many VOCs and identified conserved and non-conserved linear B-cell epitopes; ( ii ) Compared titers and neutralization antibodies specific to these conserved and non-conserved B-cell epitopes from serum of symptomatic and asymptomatic COVID-19 patients that were exposed to multiple VOCs across the 5 - year COVID-19 pandemic, and ( iii ) Compared protective efficacy of conserved versus non-conserved B-cell epitopes against the most pathogenic Delta variant in a "humanized" ACE-2/HLA transgenic mouse model. We found robust conserved B-cell epitope-specific antibody titers and neutralization in sera from asymptomatic COVID-19 patients. In contrast, sera from symptomatic patients contained weaker antibody responses specific to conserved B-cell epitopes. A multi-epitope COVID-19 vaccine that incorporated the conserved B-cell epitopes, but not the non-conserved B-cell epitopes, significantly protected the ACE2/HLA transgenic mice against infection and COVID-19 like symptoms caused by the Delta variant. These findings underscore the importance of conserved B-cell epitopes in generating robust protective immunity against severe COVID-19 symptoms caused by various VOCs, providing valuable insights for the development of broad-spectrum next generation Coronavirus vaccines capable of conferring cross-variant protective immunity., Competing Interests: Conflict of interest: Studies of this report were supported 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 and by R43AI174383 to TechImmune, LLC. LBM has an equity interest in TechImmune, LLC., a company that may potentially benefit from the research results and serves on the company’s Scientific Advisory Board. LBM’s relationship with TechImmune, LLC., has been reviewed and approved by the University of California, Irvine in accordance with its conflict-of-interest policies.

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes an asymptomatic latent infection of sensory neurons of dorsal root ganglia (DRG). Chemical and physical stress cause intermittent virus reactivation from latently infected DRG and recurrent virus shedding in the genital mucosal epithelium causing genital herpes in symptomatic patients. While T cells appear to play a role in controlling virus reactivation from DRG and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T cells into DRG and the vaginal mucosa (VM) remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4 + and CD8 + T cells and its effect on the frequency and severity of recurrent genital herpes in the recurrent herpes guinea pig model. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-2 ribonucleotide reductase 2 (RR2) protein ( Prime ) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 chemokines to recruit CD4 + and CD8 + T cells into the infected DRG and VM ( Pull ). Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident and effector memory CD4 + and CD8 + T cells in both DRG and VM tissues. This was associated with less virus in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4 + and CD8 + T cells in reducing virus shedding at the vaginal site of infection and the severity of recurrent genital herpes and propose the novel prime-pull vaccine strategy to protect against recurrent genital herpes.IMPORTANCEThe present study investigates the novel prime/pull therapeutic vaccine strategy to protect against recurrent genital herpes using the latently infected guinea pig model. In this study, we used the strategy that involves immunization of herpes simplex virus type 2-infected guinea pigs using a recombinantly expressed herpes tegument protein-ribonucleotide reductase 2 (RR2; prime), followed by intravaginal treatment with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 T-cell-attracting chemokines to recruit T cells into the infected dorsal root ganglia (DRG) and vaginal mucosa (VM) (pull). We show that the RR2/CXCL11 prime-pull therapeutic vaccine strategy elicited a significant reduction in virus shedding in the vaginal mucosa and decreased the severity and frequency of recurrent genital herpes. This protection was associated with increased frequencies of functional tissue-resident (T RM cells) and effector (T EM cells) memory CD4 + and CD8 + T cells infiltrating latently infected DRG tissues and the healed regions of the vaginal mucosa. These findings shed light on the role of tissue-resident and effector memory CD4 + and CD8 + T cells in DRG tissues and the VM in protection against recurrent genital herpes and propose the prime-pull therapeutic vaccine strategy in combating genital herpes., Competing Interests: The authors declare no conflict of interest.

Background: Cross-reactive SARS-CoV-2-specific memory CD4 + and CD8 + T cells are present in up to 50% of unexposed, pre-pandemic, healthy individuals (UPPHIs). However, the characteristics of cross-reactive memory CD4 + and CD8 + T cells associated with subsequent protection of asymptomatic coronavirus disease 2019 (COVID-19) patients (i.e., unvaccinated individuals who never develop any COVID-19 symptoms despite being infected with SARS-CoV-2) remains to be fully elucidated., Methods: This study compares the antigen specificity, frequency, phenotype, and function of cross-reactive memory CD4 + and CD8 + T cells between common cold coronaviruses (CCCs) and SARS-CoV-2. T-cell responses against genome-wide conserved epitopes were studied early in the disease course in a cohort of 147 unvaccinated COVID-19 patients who were divided into six groups based on the severity of their symptoms., Results: Compared to severely ill COVID-19 patients and patients with fatal COVID-19 outcomes, the asymptomatic COVID-19 patients displayed significantly: (i) higher rates of co-infection with the 229E alpha species of CCCs (α-CCC-229E); (ii) higher frequencies of cross-reactive functional CD134 + CD137 + CD4 + and CD134 + CD137 + CD8 + T cells that cross-recognized conserved epitopes from α-CCCs and SARS-CoV-2 structural, non-structural, and accessory proteins; and (iii) lower frequencies of CCCs/SARS-CoV-2 cross-reactive exhausted PD-1 + TIM3 + TIGIT + CTLA4 + CD4 + and PD-1 + TIM3 + TIGIT + CTLA4 + CD8 + T cells, detected both ex vivo and in vitro ., Conclusions: These findings (i) support a crucial role of functional, poly-antigenic α-CCCs/SARS-CoV-2 cross-reactive memory CD4 + and CD8 + T cells, induced following previous CCCs seasonal exposures, in protection against subsequent severe COVID-19 disease and (ii) provide critical insights into developing broadly protective, multi-antigen, CD4 + , and CD8 + T-cell-based, universal pan-Coronavirus vaccines capable of conferring cross-species protection., Competing Interests: Authors HV, DG, TJ and JU were employed by company TechImmune LLC. LB has an equity interest in TechImmune, LLC., a company that may potentially benefit from the research results and serves on the company’s Scientific Advisory Board. LB’s relationship with TechImmune, LLC., has been reviewed and approved by the University of California, Irvine by its conflict-of-interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Coulon, Prakash, Dhanushkodi, Srivastava, Zayou, Tifrea, Edwards, Figueroa, Schubl, Hsieh, Nesburn, Kuppermann, Bahraoui, Vahed, Gil, Jones, Ulmer and BenMohamed.)

SARS-CoV-2 variants of concern (VOCs) continue to evolve and reemerge with chronic inflammatory long COVID sequelae, necessitating the development of anti-inflammatory therapeutic molecules. Therapeutic effects of the receptor for advanced glycation end products (RAGE) were reported in many inflammatory diseases. However, a therapeutic effect of RAGE in COVID-19 has not been reported. In the present study, we investigated whether and how the RAGE-Ig fusion protein would have an antiviral and anti-inflammatory therapeutic effect in the COVID-19 system. The protective therapeutic effect of RAGE-Ig was determined in vivo in K18-hACE2 transgenic mice and Syrian golden hamsters infected with six VOCs of SARS-CoV-2. The underlying antiviral mechanism of RAGE-Ig was determined in vitro in SARS-CoV-2-infected human lung epithelial cells (BEAS-2B). Following treatment of K18-hACE2 mice and hamsters infected with various SARS-CoV-2 VOCs with RAGE-Ig, we demonstrated (1) significant dose-dependent protection (i.e., greater survival, less weight loss, lower virus replication in the lungs); (2) a reduction of inflammatory macrophages (F4/80+/Ly6C+) and neutrophils (CD11b+/Ly6G+) infiltrating the infected lungs; (3) a RAGE-Ig dose-dependent increase in the expression of type I IFNs (IFN-α and IFN-β) and type III IFN (IFNλ2) and a decrease in the inflammatory cytokines (IL-6 and IL-8) in SARS-CoV-2-infected human lung epithelial cells; and (4) a dose-dependent decrease in the expression of CD64 (FcgR1) on monocytes and lung epithelial cells from symptomatic COVID-19 patients. Our preclinical findings revealed type I and III IFN-mediated antiviral and anti-inflammatory therapeutic effects of RAGE-Ig protein against COVID-19 caused by multiple SARS-CoV-2 VOCs., (Copyright © 2024 by The American Association of Immunologists, Inc.)

The first-generation Spike-alone-based COVID-19 vaccines have successfully contributed to reducing the risk of hospitalization, serious illness, and death caused by SARS-CoV-2 infections. However, waning immunity induced by these vaccines failed to prevent immune escape by many variants of concern (VOCs) that emerged from 2020 to 2024, resulting in a prolonged COVID-19 pandemic. We hypothesize that a next-generation Coronavirus (CoV) vaccine incorporating highly conserved non-Spike SARS-CoV-2 antigens would confer stronger and broader cross-protective immunity against multiple VOCs. In the present study, we identified ten non-Spike antigens that are highly conserved in 8.7 million SARS-CoV-2 strains, twenty-one VOCs, SARS-CoV, MERS-CoV, Common Cold CoVs, and animal CoVs. Seven of the 10 antigens were preferentially recognized by CD8 + and CD4 + T-cells from unvaccinated asymptomatic COVID-19 patients, irrespective of VOC infection. Three out of the seven conserved non-Spike T cell antigens belong to the early expressed Replication and Transcription Complex (RTC) region, when administered to the golden Syrian hamsters, in combination with Spike, as nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP) (i.e., combined mRNA/LNP-based pan-CoV vaccine): ( i ) Induced high frequencies of lung-resident antigen-specific CXCR5 + CD4 + T follicular helper (T FH ) cells, GzmB + CD4 + and GzmB + CD8 + cytotoxic T cells (T CYT ), and CD69 + IFN-γ + TNFα + CD4 + and CD69 + IFN-γ + TNFα + CD8 + effector T cells (T EFF ); and ( ii ) Reduced viral load and COVID-19-like symptoms caused by various VOCs, including the highly pathogenic B.1.617.2 Delta variant and the highly transmittable heavily Spike-mutated XBB1.5 Omicron sub-variant. The combined mRNA/LNP-based pan-CoV vaccine could be rapidly adapted for clinical use to confer broader cross-protective immunity against emerging highly mutated and pathogenic VOCs., Competing Interests: These studies were supported in part 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 and by R43AI174383 to TechImmune, LLC. LBM has an equity interest in TechImmune, LLC., a company that may potentially benefit from the research results and serves on the company's Scientific Advisory Board. LBM's relationship with TechImmune, LLC., has been reviewed and approved by the University of California, Irvine by its conflict-of-interest policies.Studies of this report were supported 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. LBM has an equity interest in TechImmune, LLC., a company that may potentially benefit from the research results and serves on the company's Scientific Advisory Board. LBM's relationship with TechImmune, LLC., has been reviewed and approved by the University of California, Irvine by its conflict-of-interest policies.

Background: The coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has decreased significantly, the long-term outlook of COVID-19 remains a serious cause of morbidity and mortality worldwide, with the mortality rate still substantially surpassing even that recorded for influenza viruses. The continued emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, has prolonged the COVID-19 pandemic and underscores the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs., Methods: 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-variant SARS-CoV-2 vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model., Results: The pan-variant SARS-CoV-2 vaccine (i) is safe , (ii) induces high frequencies of lung-resident functional CD8 + and CD4 + T EM and T RM cells , and (iii) provides robust protection against morbidity and virus replication. COVID-19-related lung pathology and death were 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)., Conclusion: A multi-epitope pan-variant SARS-CoV-2 vaccine bearing conserved human B- and T- cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that facilitated virus clearance, and reduced morbidity, COVID-19-related lung pathology, and death caused by multiple SARS-CoV-2 VOCs., Competing Interests: LB has an equity interest in TechImmune, LLC., a company that may potentially benefit from the research results and serves on the company’s Scientific Advisory Board. LB’s relationship with TechImmune, LLC., has been reviewed and approved by the University of California, Irvine by its conflict-of-interest policies. Authors, HV, JU, DG, TJ were employed by TechImmune, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Prakash, Dhanushkodi, Zayou, Ibraim, Quadiri, Coulon, Tifrea, Suzer, Shaik, Chilukuri, Edwards, Singer, Vahed, Nesburn, Kuppermann, Ulmer, Gil, Jones and BenMohamed.)

Importance: Although the current rate of SARS-CoV-2 infections has decreased significantly, COVID-19 still ranks very high as a cause of death worldwide. As of October 2023, the weekly mortality rate is still at 600 deaths in the United States alone, which surpasses even the worst mortality rates recorded for influenza. Thus, the long-term outlook of COVID-19 is still a serious concern outlining the need for the next-generation vaccine. This study found that a prime/pull coronavirus vaccine strategy increased the frequency of functional SARS-CoV-2-specific CD4 + and CD8 + memory T cells in the lungs of SARS-CoV-2-infected triple transgenic HLA-DR*0101/HLA-A*0201/hACE2 mouse model, thereby resulting in low viral titer and reduced COVID-19-like symptoms., Competing Interests: L.B.M. has an equity interest in TechImmune, LLC, a company that may potentially benefit from the research results and serves on the company's Scientific Advisory Board. L.B.M.'s relationship with TechImmune, LLC has been reviewed and approved by the University of California, Irvine, in accordance with its conflict-of-interest policies.

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes latency in sensory neurons of the dorsal root ganglia (DRG). Intermittent virus reactivation from latency and shedding in the vaginal mucosa (VM) causes recurrent genital herpes. While T-cells appear to play a role in controlling virus reactivation and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T-cells into DRG and VM tissues remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4 + and CD8 + T cells and its effect on the frequency and severity of recurrent genital herpes. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-1 RR2 protein (Prime) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 (AAV-8) expressing CXCL9, CXCL10, or CXCL11 T-cell-attracting chemokines ( Pull) . Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident (T RM cells) and effector (T EM cells) memory CD4 + and CD8 + T cells in both DRG and VM tissues. This was associated with less virus shedding in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4 + and CD8 + T RM and T EM cells in reducing virus reactivation shedding and the severity of recurrent genital herpes and propose the novel prime/pull vaccine strategy to protect against recurrent genital herpes.

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., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Vaginal mucosa-resident anti-viral effector memory B- and T cells appeared to play a crucial role in protection against genital herpes. However, how to mobilize such protective immune cells into the vaginal tissue close to infected epithelial cells remains to be determined. In the present study, we investigate whether and how, CCL28, a major mucosal-associated chemokine, mobilizes effector memory B- and T cells in leading to protecting mucosal surfaces from herpes infection and disease. The CCL28 is a chemoattractant for the CCR10 receptor-expressing immune cells and is produced homeostatically in the human vaginal mucosa (VM). We found the presence of significant frequencies of HSV-specific memory CCR10 + CD44 + CD8 + T cells, expressing high levels of CCR10 receptor, in herpes-infected asymptomatic (ASYMP) women compared to symptomatic (SYMP) women. A significant amount of the CCL28 chemokine (a ligand of CCR10), was detected in the VM of herpes-infected ASYMP B6 mice, associated with the mobilization of high frequencies of HSV-specific effector memory CCR10 + CD44 + CD62L - CD8 + T EM cells and memory CCR10 + B220 + CD27 + B cells in the VM of HSV-infected asymptomatic mice. In contrast, compared to wild-type (WT) B6 mice, the CCL28 knockout (CCL28 (-/-) ) mice: ( i ) Appeared more susceptible to intravaginal infection and re-infection with HSV-2; ( ii ) Exhibited a significant decrease in the frequencies of HSV-specific effector memory CCR10 + CD44 + CD62L - CD8 + T EM cells and of memory CD27 + B220 + B cells in the infected VM. The results imply a critical role of the CCL28/CCR10 chemokine axis in the mobilization of anti-viral memory B and T cells within the VM to protect against genital herpes infection and disease., Competing Interests: Conflict of interest: The authors have declared that no conflicts of interest exist.

Background: The 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., Methods: In 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., Results: The Pan-Coronavirus vaccine: ( i ) is safe; ( ii ) induces high frequencies of lung-resident functional CD8 + and CD4 + T EM and T RM cells; 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., Competing Interests: Declaration of Interest: The University of California Irvine has filed a patent application on the results reported in this manuscript.

Mutations in ATP13A2 ( PARK9 ), an autophagy-related protein, cause Kufor-Rakeb syndrome, an autosomal recessive, juvenile-onset form of parkinsonism. α-Synuclein (α-syn) is a presynaptic neuronal protein that forms toxic aggregates in Parkinson's disease (PD). We studied α-syn aggregation and autophagic flux in ATP13A2-knockdown Drosophila expressing either wild-type (WT) or mutant α-syn. Dopaminergic (DA) neuron loss was studied by confocal microscopy. Sleep and circadian activity were evaluated in young and old flies using a Drosophila activity monitor. Thirty-day-old ATP13A2 -RNAi A53T-α-syn flies had increased Triton-insoluble α-syn levels, compared to control A53T-α-syn flies without ATP13A2 -RNAi. Whole-brain staining revealed significantly fewer dopaminergic (DA) neurons in the PPL2 cluster of 30-day-old ATP13A2 -RNAi flies expressing WT-, A30P-, and A53T-α-syn than in that of controls. In ATP13A2 -RNAi A53T-α-syn flies, autophagic flux was decreased, as indicated by increased accumulation of Ref(2)P, the Drosophila p62 homologue. ATP13A2 silencing decreased total locomotor activity in young, and enhanced sleep features, similar to PD (decreasing bout length), in old flies expressing A53T-α-syn. ATP13A2 silencing also altered the circadian locomotor activity of A30P- and A53T-α-syn flies. Thus, ATP13A2 may play a role in the autophagic degradation of A53T-α-syn.

The development of vaccines against herpes simplex virus type 1 and type 2 (HSV1 and HSV-2) is an important goal for global health. In this review we reexamined ( i ) the status of ocular herpes vaccines in clinical trials; and ( ii ) discusses the recent scientific advances in the understanding of differential immune response between HSV infected asymptomatic and symptomatic individuals that form the basis for the new combinatorial vaccine strategies targeting HSV; and ( iii ) shed light on our novel "asymptomatic" herpes approach based on protective immune mechanisms in seropositive asymptomatic individuals who are "naturally" protected from recurrent herpetic diseases. We previously reported that phenotypically and functionally distinct HSV-specific memory CD8 + T cell subsets in asymptomatic and symptomatic HSV-infected individuals. Moreover, a better protection induced following a prime/pull vaccine approach that consists of first priming anti-viral effector memory T cells systemically and then pulling them to the sites of virus reactivation (e.g., sensory ganglia) and replication (e.g., eyes and vaginal mucosa), following mucosal administration of vectors expressing T cell-attracting chemokines. In addition, we reported that a combination of prime/pull vaccine approach with approaches to reverse T cell exhaustion led to even better protection against herpes infection and disease. Blocking PD-1, LAG-3, TIGIT and/or TIM-3 immune checkpoint pathways helped in restoring the function of antiviral HSV-specific CD8 + T cells in latently infected ganglia and increased efficacy and longevity of the prime/pull herpes vaccine. We discussed that a prime/pull vaccine strategy that use of asymptomatic epitopes, combined with immune checkpoint blockade would prove to be a successful herpes vaccine approach., Competing Interests: Author HV was employed by TechImmune, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chentoufi, Dhanushkodi, Srivastava, Prakash, Coulon, Zayou, Vahed, Chentoufi, Hormi-Carver and BenMohamed.)

Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity seen in asymptomatic (ASYMP) individuals is heavily explored, the role of B cells is less investigated. In the present study, we evaluated whether B cells are associated with protective immunity against recurrent ocular herpes. The frequencies of circulating HSV-specific memory B cells and of memory follicular helper T cells (CD4 + T fh cells), which help B cells produce antibodies, were compared between HSV-1-infected SYMP and ASYMP individuals. The levels of IgG/IgA and neutralizing antibodies were compared in SYMP and ASYMP individuals. We found that (i) the ASYMP individuals had increased frequencies of HSV-specific CD19 + CD27 + memory B cells, and (ii) high frequencies of HSV-specific switched IgG + CD19 + CD27 + memory B cells detected in ASYMP individuals were directly proportional to high frequencies of CD45R0 + CXCR5 + CD4 + memory T fh cells. However, no differences were detected in the level of HSV-specific IgG/IgA antibodies in SYMP and ASYMP individuals. Using the UV-B-induced HSV-1 reactivation mouse model, we found increased frequencies of HSV-specific antibody-secreting plasma HSV-1 gD + CD138 + B cells within the TG and circulation of ASYMP mice compared to those of SYMP mice. In contrast, no significant differences in the frequencies of B cells were found in the cornea, spleen, and bone-marrow. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from symptomatic recurrent ocular herpes. IMPORTANCE Reactivation of herpes simplex virus 1 (HSV-1) from latently infected neurons of the trigeminal ganglia (TG) leads to blinding recurrent herpetic disease in symptomatic (SYMP) individuals. Although the role of T cells in herpes immunity against blinding recurrent herpetic disease is heavily explored, the role of B cells is less investigated. In the present study, we found that in both asymptomatic (ASYMP) individuals and ASYMP mice, there were increased frequencies of HSV-specific memory B cells that were directly proportional to high frequencies of memory T fh cells. Moreover, following UV-B-induced reactivation, we found increased frequencies of HSV-specific antibody-secreting plasma B cells within the TG and circulation of ASYMP mice compared to those of SYMP mice. Our findings suggest that circulating antibody-producing HSV-specific memory B cells recruited locally to the TG may contribute to protection from recurrent ocular herpes.

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., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Herpes simplex virus 1 (HSV-1) infects the cornea and caused blinding ocular disease. In the present study, we evaluated whether and how a novel engineered version of fibroblast growth factor-1 (FGF-1), designated as TTHX1114, would reduce the severity of HSV-1-induced and recurrent ocular herpes in the mouse model. The efficacy of TTHX1114 against corneal keratopathy was assessed in B6 mice following corneal infection with HSV-1, strain McKrae. Starting day one post infection (PI), mice received TTHX1114 for 14 days. The severity of primary stromal keratitis and blepharitis were monitored up to 28 days PI. Inflammatory cell infiltrating infected corneas were characterized up to day 21 PI. The severity of recurrent herpetic disease was quantified in latently infected B6 mice up to 30 days post-UVB corneal exposure. The effect of TTHX1114 on M1 and M2 macrophage polarization was determined in vivo in mice and in vitro on primary human monocytes-derived macrophages. Compared to HSV-1 infected non-treated mice, the infected and TTHX1114 treated mice exhibited significant reduction of primary and recurrent stromal keratitis and blepharitis, without affecting virus corneal replication. The therapeutic effect of TTHX1114 was associated with a significant decrease in the frequency of M1 macrophages infiltrating the cornea, which expressed significantly lower levels of pro-inflammatory cytokines and chemokines. This polarization toward M2 phenotype was confirmed in vitro on human primary macrophages. This pre-clinical finding suggests use of this engineered FGF-1 as a novel immunotherapeutic regimen to reduce primary and recurrent HSV-1-induced corneal disease in the clinic., Competing Interests: ED and DB are employees and hold an equity interest in Trefoil Therapeutics, Inc. ED is an inventor on patents claiming TTHX1114. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dhanushkodi, Srivastava, Coulon, Prakash, Roy, Bagnol, David and BenMohamed.)

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.

The nature of antiviral CD8 + T cells associated with protective and pathogenic herpes simplex virus type 1 (HSV-1) infections remains unclear. We compared the transcriptome, phenotype, and function of memory CD8 + T cells, sharing the same HSV-1 epitope-specificities, from infected HLA-A*0201 positive symptomatic (SYMP) vs. asymptomatic (ASYMP) individuals and HLA-A*0201 transgenic rabbits. Compared to higher frequencies of multifunctional effector memory CD8 + T EM cells in ASYMP individuals, the SYMP individuals presented dysfunctional CD8 + T EM cells, expressing major exhaustion pathways. Compared to protected ASYMP HLA transgenic rabbits, the trigeminal ganglia of non-protected SYMP HLA transgenic rabbits had higher frequencies of dysfunctional tissue-resident CD8 + T RM cells. Moreover, blockade of T cell exhaustion pathways restored the function of CD8 + T cells, reduced virus reactivation, and diminished recurrent disease in HLA transgenic rabbits. These findings reveal unique molecular signatures of protective CD8 + T cells and pave the way for T-cell-based immunotherapy to combat recurrent ocular herpes.

Invariant natural killer (iNKT) cells are among the first innate immune cells to elicit early protective immunity that controls invading viral pathogens. The role of the iNKT cell subsets iNKT1, iNKT2, and iNKT17 in herpesvirus immunity remains to be fully elucidated. In this study, we examined the protective role of cornea-resident iNKT cell subsets using the mouse model of ocular herpesvirus infection and disease. Wild-type (WT) C57BL/6 (B6) mice and CD1d knockout (KO) mice were infected ocularly with herpes simplex virus 1 (HSV-1) (strain McKrae). Cornea, spleen, and liver were harvested at 0, 2, 5, 8, and 14 days postinfection (p.i.), and the frequency and function of the three major iNKT cell subsets were analyzed and correlated with symptomatic and asymptomatic corneal herpesvirus infections. The profiles of 16 major pro- and anti-inflammatory cytokines were analyzed in corneal lysates using Western blot and Luminex assays. Early during ocular herpesvirus infection (i.e., day 2), the gamma interferon (IFN-γ)-producing PLZF lo RORγt lo (promyelocytic leukemia zinc finger, retinoic acid-related orphan receptor gT) iNKT1 cell subset was the predominant iNKT cell subset in infected asymptomatic corneas. Moreover, compared to the asymptomatic corneas of HSV-1-infected WT mice, the symptomatic corneas CD1d KO mice, with iNKT cell deficiency, had increased levels of the inflammatory cytokine interleukin-6 (IL-6) and decreased levels of IL-12, IFN-γ, and the JAK1, STAT1, NF-κB, and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways. Our findings suggest that IFN-γ-producing PLZF lo RORγt lo iNKT1 cells play a role in the protective innate immune response against symptomatic ocular herpes. IMPORTANCE We investigated the protective role of iNKT cell subsets in asymptomatic ocular herpesvirus infection. We found that early during ocular herpesvirus infection (i.e., on day 2 postinfection), IFN-γ-producing PLZF lo RORγt lo iNKT1 cells were the predominant iNKT cell subset in infected corneas of asymptomatic B6 mice (with little to no corneal herpetic disease), compared to corneas of symptomatic mice (with severe corneal herpetic disease). Moreover, compared to asymptomatic corneas of wild-type (WT) B6 mice, the symptomatic corneas of CD1d KO mice, which lack iNKT cells, showed (i) decreases in the levels of IFN-γ and IL-12, (ii) an increase in the level of the inflammatory cytokine IL-6; and (iii) downregulation of the JAK1, STAT1, NF-κB, and ERK1/2 pathways. The findings suggest that early during ocular herpesvirus infection, cornea-resident IFN-γ-producing PLZF lo RORγt lo iNKT1 cells provide protection from symptomatic ocular herpes., (Copyright © 2020 American Society for Microbiology.)

While the role of CD8 + T cells in the control of herpes simplex virus 1 (HSV-1) infection and disease is gaining wider acceptance, a direct involvement of effector CD4 + T cells in this protection and the phenotype and function of HSV-specific human CD4 + T cell epitopes remain to be fully elucidated. In the present study, we report that several epitopes from the HSV-1 virion tegument protein (VP11/12) encoded by UL46 are targeted by CD4 + T cells from HSV-seropositive asymptomatic individuals (who, despite being infected, never develop any recurrent herpetic disease). Among these, we identified two immunodominant effector memory CD4 + T EM cell epitopes, amino acids (aa) 129 to 143 of VP11/12 (VP11/12 129-143 ) and VP11/12 483-497 , using in silico , in vitro , and in vivo approaches based on the following: (i) a combination of the TEPITOPE algorithm and PepScan library scanning of the entire 718 aa of HSV-1 VP11/12 sequence; (ii) an in silico peptide-protein docking analysis and in vitro binding assay that identify epitopes with high affinity to soluble HLA-DRB1 molecules; and (iii) an ELISpot assay and intracellular detection of gamma interferon (IFN-γ), CD107 a/b degranulation, and CD4 + T cell carboxyfluorescein succinimidyl ester (CFSE) proliferation assays. We demonstrated that native VP11/12 129-143 and VP11/12 483-497 epitopes presented by HSV-1-infected HLA-DR-positive target cells were recognized mainly by effector memory CD4 + T EM cells while being less targeted by FOXP3 + CD4 + CD25 + regulatory T cells. Furthermore, immunization of HLA-DR transgenic mice with a mixture of the two immunodominant human VP11/12 CD4 + T EM cell epitopes, but not with cryptic epitopes, induced HSV-specific polyfunctional IFN-γ-producing CD107 ab+ CD4 + T cells associated with protective immunity against ocular herpes infection and disease. IMPORTANCE We report that naturally protected HSV-1-seropositive asymptomatic individuals develop a higher frequency of antiviral effector memory CD4 + T EM cells specific to two immunodominant epitopes derived from the HSV-1 tegument protein VP11/12. Immunization of HLA-DR transgenic mice with a mixture of these two immunodominant CD4 + T cell epitopes induced a robust antiviral CD4 + T cell response in the cornea that was associated with protective immunity against ocular herpes. The emerging concept of developing an asymptomatic herpes vaccine that would boost effector memory CD4 + and CD8 + T EM cell responses is discussed., (Copyright © 2020 American Society for Microbiology.)

Background: Mutations in glucocerebrosidase (GBA), a lysosomal enzyme are the most common genetic risk factor for developing Parkinson's disease (PD). We studied how reduced GCase activity affects α-synuclein (α-syn) and its mutants (A30P and A53T) aggregation, neurodegeneration, sleep and locomotor behavior in a fly model of PD. Methods: We developed drosophila with GBA gene knockdown (RNAi) (with reduced GCase activity) that simultaneously expresses either wildtype (WT) or mutants such as A30P or A53T α-syn. Western blot and confocal microscopy were performed to study the α-syn aggregation and neurodegeneration in these flies. We also studied the sleep and locomotor activity of those flies using Drosophila activity monitor (DAM) system. Results: Western blot analysis showed that GBA RNAi A53T α-syn flies (30 days old) had an increased level of Triton insoluble synuclein (that corresponds to α-syn aggregates) compared to corresponding A53T flies without GBA RNAi (control), while mRNA expression of α-syn remained unchanged. Confocal imaging of whole brain staining of 30 days old drosophila showed a statistically significant decrease in neuron numbers in PPL1 cluster in flies expressing α-syn WT, A30P and A53T in the presence GBA RNAi compared to corresponding control. Staining with conformation specific antibody for α-syn aggregates showed an increased number of neurons staining for α-syn aggregates in A53T fly brain with GBA RNAi compared to control A53T flies, thus confirming our protein analysis finding that under decreased GBA enzyme activity, mutant A53T aggregates more than the control A53T without GBA silencing. Sleep analysis revealed decreased total activity in GBA silenced flies expressing mutant A53T compared to both A53T control flies and GBA RNAi flies without synuclein expression. Conclusion: In A53T flies with reduced GCase activity, there is increased α-syn aggregation and dopamine (DA) neuronal loss. This study demonstrates that reduced GCase activity both in the context of heterozygous GBA1 mutation associated with PD and in old age, contribute to increased aggregation of mutant α-syn A53T and exacerbates the phenotype in a fly model of PD.

The mammalian target of rapamycin (mTOR) plays critical roles in immunity. We previously showed that infection of human embryonic kidney (HEK) cells with Sindbis virus (SIN), an enveloped RNA alphavirus, profoundly suppresses Akt/mTOR signaling, and host translation late during infection. To understand how SIN mediated suppression of mTOR affects innate response, we analyzed phosphorylation of interferon regulatory factor 3 (IRF3) and expression of two antiviral genes. Here we show strong phosphorylation of IRF3, and an increase in mRNA levels for antiviral genes interferon stimulated gene (ISG)56 and interferon gamma inducible protein (IP)-10 when intracellular viral RNA levels are high during late infection. The mTOR inhibitors rapamycin and torin1 do not block, but mildly upregulate these responses. Even after prolonged treatment with Ly294002, the PI3K inhibitor only partially blocks SIN induced phosphorylation of IRF3. While Ly294002 treatment downregulated the SIN induced expression of ISG56 mRNA levels, it had no effect on SIN induced upregulation of IP-10 expression. These results point to SIN replication-mediated activation of IRF3, independent of mTOR function, when host protein synthesis is severely suppressed by virus infection.

Chemokines play a pivotal role in the innate response to both bacterial and viral infections, and in mixed infections. To determine chemokine responses to Sindbis virus (SIN) in a co-infection model, peripheral blood mononuclear cells (PBMCs) derived from healthy volunteers were exposed to SIN in the presence and absence of lipopolysaccharide (LPS). Culture supernatants recovered at 2, 24, and 72 h post-exposure were evaluated for virus replication and analyzed for chemokines by ELISA. None of the PBMC cultures showed new virus release, GFP reporter expression, or viral RNA synthesis. While SIN had little effect on the induction of IL-8 and RANTES, the chemokines MCP-1, MIP1-α (p < 0.001), and MIP1-β (p < 0.0004) were drastically upregulated by SIN as well as LPS. Both live and UV-inactivated SIN induced secretion of IP-10 and I-TAC. Although LPS did not induce release of IP-10, it sharply inhibited (p = 0.004) SIN-mediated IP-10 secretion. On the contrary, the release of SLC was blocked by SIN. The adjuvant activity of IP-10, its antiangiogenic function, and antagonism between SIN and LPS for the release of select chemokines may be useful in understanding the pathogenesis of mixed infections, cross-talk between cellular pathways, and may have applications in cancer and sepsis.

Genetically engineered Sindbis viruses (SIN) are excellent oncolytic agents in preclinical models. Several human cancers have aberrant Akt signaling, and kinase inhibitors including rapamycin are currently tested in combination therapies with oncolytic viruses. Therefore, it was of interest to delineate possible cross-regulation between SIN replication and PI3K/Akt/mTOR signaling. Here, using HEK293T cells as host, we report the following key findings: (a) robust SIN replication occurs in the presence of mTOR specific inhibitors, rapamycin and torin1 or Ly294002--a PI3K inhibitor, suggesting a lack of requirement for PI3K/Akt/mTOR signaling; (b) suppression of phosphorylation of Akt, mTOR and its effectors S6, and 4E-BP1 occurs late during SIN infection: a viral function that may be beneficial in counteracting cellular drug resistance to kinase inhibitors; (c) Ly294002 and SIN act additively to suppress PI3K/Akt/mTOR pathway with little effect on virus release; and (d) SIN replication induces host translational shut off, phosphorylation of eIF2α and apoptosis. This first report on the potent inhibition of Akt/mTOR signaling by SIN replication, bolsters further studies on the development and evaluation of engineered SIN genotypes in vitro and in vivo for unique cytolytic functions., (Copyright © 2011 Elsevier Inc. All rights reserved.)