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The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of the recombinant RBD produced in different mammalian cells. We found a higher degree of complex-type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated, and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity, while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity, verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it is possible to enhance the immunogenicity of recombinant viral proteins.

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Human cytomegalovirus (CMV) infection is reported to promote NK cell differentiation and education. The CMV-induced generation of highly differentiated adaptive-like NK cells has been proposed to affect favorably on the maintenance of remission in patients with acute myeloid leukemia (AML) after allogeneic stem cell transplantation (allo-SCT). The impact of CMV infection and adaptive-like NK cells on relapse and survival of patients with AML not receiving allo-SCT remains unknown. We assayed CMV IgG serostatus to determine past CMV infection in 81 nontransplanted AML patients who were receiving relapse-prevention immunotherapy comprising histamine dihydrochloride and low-dose interleukin-2 (HDC/IL2; NCT01347996). CMV seropositivity correlated negatively with leukemia-free and overall survival of patients receiving HDC/IL2, but did not correlate with outcomes in a contemporary control cohort. Analysis of outcome after stratification of patients based on concordant or discordant killer immunoglobulin-like receptor (KIR) and HLA genotypes implied that the negative impact of CMV seropositivity was restricted to patients lacking a ligand to inhibitory KIRs (iKIR). Previous CMV infection was also associated with fewer NK cells expressing only nonself iKIRs (NS-iKIR). We propose that CMV-driven NK cell education depletes the population of NS-iKIR NK cells, which in turn reduces the clinical benefit of relapse-preventive immunotherapy in AML. Cancer Immunol Res; 6(9); 1110–9. ©2018 AACR.

Solid organ transplant recipients (SOTRs) are on lifelong immunosuppression, which may interfere with adaptive immunity to COVID-19. The data on dynamics and duration of antibody response in SOTRs are limited. This longitudinal study examined the longevity of both anti-spike (S)- and anti-nucleocapsid (N)-specific IgG antibodies after COVID-19 in SOTRs compared to matched immunocompetent persons. SOTRs (n = 65) were matched with controls (n = 65) for COVID-19 disease severity, age, and sex in order of priority. Serum-IgG antibodies against N and S antigens of SARS-CoV-2 were analyzed. At 1 and 9 months after COVID-19, anti-S-IgG detectability decreased from 91% to 82% in SOTRs versus 100% to 95% in controls, whereas the anti-N-IgG decreased from 63% to 29% in SOTRs versus 89% to 46% in controls. A matched paired analysis showed SOTRs having significantly lower levels of anti-N-IgG at all time points (1 month p = .007, 3 months p .001, 6 months p = .019, and 9 months p = .021) but not anti-S-IgG at any time points. A mixed-model analysis confirmed these findings except for anti-S-IgG at 1 month (p = .005) and identified severity score as the most important predictor of antibody response. SOTRs mount comparable S-specific, but not N-specific, antibody responses to SARS-CoV-2 infection compared to immunocompetent controls.

Solid organ transplant (SOT) recipients run a high risk for adverse outcomes from COVID-19, with reported mortality around 19%. We retrospectively reviewed all known Swedish SOT recipients with RT-PCR confirmed COVID-19 between March 1 and November 20, 2020 and analyzed patient characteristics, management, and outcome. We identified 230 patients with a median age of 54.0 years (13.2), who were predominantly male (64%). Most patients were hospitalized (64%), but 36% remained outpatients. Age >50 and male sex were among predictors of transition from outpatient to inpatient status. National early warning Score 2 (NEWS2) at presentation was higher in non-survivors. Thirty-day all-cause mortality was 9.6% (15.0% for inpatients), increased with age and BMI, and was higher in men. Renal function decreased during COVID-19 but recovered in most patients. SARS-CoV-2 antibodies were identified in 78% of patients at 1-2 months post-infection. Nucleocapsid-specific antibodies decreased to 38% after 6-7 months, while spike-specific antibody responses were more durable. Seroprevalence in 559 asymptomatic patients was 1.4%. Many patients can be managed on an outpatient basis aided by risk stratification with age, sex, and NEWS2 score. Factors associated with adverse outcomes include older age, male sex, greater BMI, and a higher NEWS2 score.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of recombinant RBD produced in different mammalian cells. We found a higher degree of complex type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in Human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it might be possible to enhance the immunogenicity of recombinant viral proteins.

Herpes simplex virus type 2 (HSV-2) is a common sexually transmitted disease that affects approximately 500 million individuals globally. There is currently no approved vaccine to prevent HSV-2 infection. EXCT4 is a truncated form of the mature glycoprotein G-2 (mgG-2) that unlike full mature form is secreted by expressing cells enabling it to be rapidly scaled up for production. The current study examined whether EXCT4 immunity in mice could be further enhanced through use of adjuvants. EXCT4 formulated with Advax-CpG adjuvant induced a strong Th1-type immune response characterized by interferon gamma (IFN-γ) and protected animals against a lethal genital challenge with HSV-2. This response was associated with reduced viral load in vaginal washes, spinal cord, and dorsal root ganglia. Together the results provide proof of concept that EXCT4 formulated with Advax-CpG adjuvant is a promising HSV-2 vaccine candidate warranting further investigation.

Background Parvovirus B19 (PVB19) has emerged as one of the viruses possibly inducing chronic myocarditis and subsequent idiopathic dilated cardiomyopathy (IDCM). The aim of this work was to investigate the presence and long-term consequences of PVB19-DNA within myocardial biopsies from patients with IDCM and to compare the findings with those from donor hearts (control group). Methods and Results Forty hospitalized IDCM patients (age 47 ± 12 y) with mean left ventricular ejection fraction 27 ± 12% were included. The presence of PVB19-DNA in myocardial biopsies and of IgG and IgM antibodies in patient sera was analyzed. The control group consisted of 20 donor hearts. The follow-up time was 112 ± 57 months. PVB19-DNA was found in myocardial biopsies of both patients (73%) and control samples (55%; P = .25).Three deaths and 8 heart transplantations occurred in the IDCM group, and 6 deaths in the control group (ie, the recipients of the control hearts). No difference in transplantation-free survival between the PVB19-DNA positive/negative IDCM patients or transplant recipients was found. Conclusions PVB19-DNA is a common finding in both patients with IDCM and in healthy donor hearts, not affecting prognosis. These findings support the view that PVB19 is an innocent bystander, frequently found in myocardium with low DNA copies, and not a plausible cause of IDCM.

The contribution of virus components to liberation of herpes simplex virus type 2 (HSV-2) progeny virions from the surface of infected cells is poorly understood. We report that the HSV-2 mutant deficient in the expression of a mucin-like membrane-associated glycoprotein G (mgG) exhibited defect in the release of progeny virions from infected cells manifested by ~2 orders of magnitude decreased amount of infectious virus in a culture medium as compared to native HSV-2. Electron microscopy revealed that the mgG deficient virions were produced in infected cells and present at the cell surface. These virions could be forcibly liberated to a nearly native HSV-2 level by the treatment of cells with glycosaminoglycan (GAG)-mimicking oligosaccharides. Comparative assessment of the interaction of mutant and native virions with surface-immobilized chondroitin sulfate GAG chains revealed that while the mutant virions associated with GAGs ~fourfold more extensively, the lateral mobility of bound virions was much poorer than that of native virions. These data indicate that the mgG of HSV-2 balances the virus interaction with GAG chains, a feature critical to prevent trapping of the progeny virions at the surface of infected cells.

Background Bolivia has the highest prevalence of cervical cancer in South America and the prevalence of viral sexually transmitted infections (STIs) among people in urban cities is increasing. Little is known about the prevalence of viral STIs in rural communities, which generally have limited access to health care. In order to study the prevalence of viral STIs in rural Bolivia, we recruited women from villages and towns in the Department of La Paz in Bolivia. Methods Three hundred ninety-four female participants were assessed for IgG-antibodies to herpes simplex virus type 2 (HSV-2), human immunodeficiency virus (HIV) and hepatitis B virus (HBV, anti-HBc), as well as for the presence of HBV surface antigen (HBsAg) in dried blood spots. The prevalence of 12 high-risk types of human papillomavirus (HPV) was assessed by qPCR in dried cervicovaginal cell spots from 376 of these women. χ2 test was used to compare variables between the populations and binary logistic regression was used to identify risk factors associated with the positivity of the tests. Results The seroprevalence of HSV-2 was 53% and of HBV 10.3%. HBAg was detected in 15.8% of women with anti-HBV antibodies indicating chronic infection. The frequency of high-risk HPV infection was 27%, with the most prevalent high-risk HPV types being HPV 56, 39 and 31 followed by HPV 16 and 18. Finally, none of the 394 women were seropositive for HIV, and about 64% of the studied population was positive for at least one of the viral infections. Conclusions Women in Bolivian rural communities in La Paz show a high prevalence of HBV, HPV and, in particular, HSV-2. In contrast, none of the women were HIV positive, suggesting that the HIV prevalence in this population is low. The pattern of high-risk HPV types differed from many other countries with a predominance of HPV-types not included in the Gardasil vaccine which was officially introduced in Bolivia in April 2017.

Human cytomegalovirus (CMV) infection is reported to promote NK cell differentiation and education. The CMV-induced generation of highly differentiated adaptive-like NK cells has been proposed to affect favorably on the maintenance of remission in patients with acute myeloid leukemia (AML) after allogeneic stem cell transplantation (allo-SCT). The impact of CMV infection and adaptive-like NK cells on relapse and survival of patients with AML not receiving allo-SCT remains unknown. We assayed CMV IgG serostatus to determine past CMV infection in 81 nontransplanted AML patients who were receiving relapse-prevention immunotherapy comprising histamine dihydrochloride and low-dose interleukin-2 (HDC/IL2; NCT01347996). CMV seropositivity correlated negatively with leukemia-free and overall survival of patients receiving HDC/IL2, but did not correlate with outcomes in a contemporary control cohort. Analysis of outcome after stratification of patients based on concordant or discordant killer immunoglobulin-like receptor (KIR) and HLA genotypes implied that the negative impact of CMV seropositivity was restricted to patients lacking a ligand to inhibitory KIRs (iKIR). Previous CMV infection was also associated with fewer NK cells expressing only nonself iKIRs (NS-iKIR). We propose that CMV-driven NK cell education depletes the population of NS-iKIR NK cells, which in turn reduces the clinical benefit of relapse-preventive immunotherapy in AML. Cancer Immunol Res; 6(9); 1110–9. ©2018 AACR.

Background: Bolivia has the highest prevalence of cervical cancer in South America and the prevalence of viral sexually transmitted infections (STIs) among people in urban cities is increasing. Little is known about the prevalence of viral STIs in rural communities, which generally have limited access to health care. In order to study the prevalence of viral STIs in rural Bolivia, we recruited women from villages and towns in the Department of La Paz in Bolivia. Methods: 394 female participants were assessed for IgG-antibodies to herpes simplex virus type 2 (HSV-2), human immunodeficiency virus (HIV) and hepatitis B virus (HBV, anti-HBc), as well as for the presence of HBV surface antigen (HBsAg) in dried blood spots. The prevalence of 12 high-risk types of human papillomavirus (HPV) was assessed by qPCR in dried cervicovaginal cell spots from 376 of these women. χ 2 test was used to compare variables between the populations and binary logistic regression was used to identify risk factors associated with the positivity of the tests. Results: The seroprevalence of HSV-2 was 53% and of HBV 10.3%. HBAg was detected in 15.8% of women with anti-HBV antibodies indicating chronic infection. The frequency of high-risk HPV infection was 27%, with the most prevalent high-risk HPV types being HPV 56, 39 and 31 followed by HPV 16 and 18. Finally, none of the 394 women were seropositive for HIV, and about 64% of the studied population was positive for at least one of the viral infections. Conclusions: Women in Bolivian rural communities in La Paz show a high prevalence of HBV, HPV and, in particular, HSV-2, but a low prevalence of HIV. The pattern of high-risk HPV types differed from e.g. Sweden with a predominance of HPV-types not included in the Gardasil vaccine which was officially introduced in Bolivia in April 2017.

In an outbreak of measles in Gothenburg, Sweden, breakthrough infections (i.e. infections in individuals with a history of vaccination) were common. The objective of this study was to compare measles RNA levels between naïve (i.e. primary) and breakthrough infections. We also propose a fast provisional classification of breakthrough infections. Medical records were reviewed and real-time PCR-positive samples genotyped. Cases were classified as naïve, breakthrough or vaccine infections. We compared clinical symptoms and measles RNA cycle threshold (Ct) values between breakthrough and naïve infections. Sixteen of 28 confirmed cases of measles in this outbreak were breakthrough infections. A fast provisional classification, based on previous history of measles vaccination and detectable levels of measles IgG in acute serum, correctly identified 14 of the 16 breakthrough infections, confirmed by IgG avidity testing. Measles viral load was significantly lower in nasopharyngeal samples from individuals with breakthrough compared with naïve infections (median Ct-values: 32 and 19, respectively, p

Herpes simplex virus (HSV) and many other viruses, including HIV, initiate infection of host cells by binding to glycosaminoglycan (GAG) chains of cell surface proteoglycans. Although GAG mimetics, such as sulfated oligo- and polysaccharides, exhibit potent antiviral activities in cultured cells, the prophylactic application of these inhibitors as vaginal microbicides failed to protect women upon their exposure to HIV. A possible explanation for this failure is that sulfated oligo- and polysaccharides exhibit no typical virucidal activity, as their interaction with viral particles is largely electrostatic and reversible and thereby vulnerable to competition with GAG-binding proteins of the genital tract. Here we report that the cholestanol-conjugated sulfated oligosaccharide PG545, but not several other sulfated oligosaccharides lacking this modification, exhibited virucidal activity manifested as disruption of the lipid envelope of HSV-2 particles. The significance of the virus particle-disrupting activity of PG545 was also demonstrated in experimental animals, as this compound, in contrast to unmodified sulfated oligosaccharide, protected mice against genital infection with HSV-2. Thus, PG545 offers a novel prophylaxis option against infections caused by GAG-binding viruses.

BackgroundTo accurately interpret COVID-19 seroprevalence surveys, knowledge of serum-IgG responses to SARS-CoV-2 with a better understanding of patients who do not seroconvert, is imperative. This study aimed to describe serum-IgG responses to SARS-CoV-2 in a cohort of patients with both severe and mild COVID-19, including extended studies of patients who remained seronegative more than 90 days post symptom onset.ResultsForty-seven patients (mean age 49 years, 38% female) were included. All (15/15) patients with severe symptoms and 29/32 (90.6%) patients with mild symptoms of COVID-19 developed SARS-CoV-2-specific IgG antibodies in serum. Time to seroconversion was significantly shorter (median 11 vs. 22 days, P=0.04) in patients with severe compared to mild symptoms. Of the three patients without detectable IgG-responses after >90 days, all had detectable virus-neutralizing antibodies and in two, spike-protein receptor binding domain-specific IgG was detected with an in-house assay. Antibody titers were preserved during follow-up and all patients who seroconverted, irrespective of the severity of symptoms, still had detectable IgG levels >75 days post symptom onset.ConclusionsPatients with severe COVID-19 both seroconvert earlier and develop higher concentrations of SARS-CoV-2-specific IgG than patients with mild symptoms. Of those patients who not develop detectable IgG antibodies, all have detectable virus-neutralizing antibodies, suggesting immunity. Our results showing that not all COVID-19 patients develop detectable IgG using two validated commercial clinical methods, even over time, are vital for the interpretation of COVID-19 seroprevalence surveys and for estimating the true infection prevalence in populations.

We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2) of herpes simplex virus 2 (HSV-2) in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1) and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (ACMC) activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials.

IFN-γ–inducible protein 16 (IFI16) is an immunological DNA sensor proposed to act in the cyclic GMP–AMP synthase–stimulator of IFN genes pathway. Because mice do not have a clear ortholog of IFI16, this system is not suitable for genetic studies of IFI16. In this study, we have compared the dependency on IFI16, cyclic GMP–AMP synthase, and stimulator of IFN genes for type I IFN induction by a panel of pathogenic bacteria and DNA viruses. The IFN response induced by HSV-2 was particularly dependent on IFI16. In a cohort of patients with genital herpes and healthy controls, the minor G allele of the IFI16 single nucleotide polymorphism rs2276404 was associated with resistance to infection. Furthermore, the combination of this allele with the C allele of rs1417806 was significantly overrepresented in uninfected individuals. Cells from individuals with the protective GC haplotype expressed higher levels of IFI16 and induced more IFN-β upon HSV-2 infection. These data provide genetic evidence for a role for IFI16 in protection against genital herpes.

Objectives During the last decades, a changing epidemiological pattern of genital herpes simplex virus (HSV) infection has emerged. Primary infection is now caused as often by HSV-1 as by HSV-2. Once established, HSV can be reactivated leading to recurrent mucocutaneous lesions as well as meningitis. Why some otherwise immune-competent individuals experience severe and frequent recurrences is not known, and the immunological mechanism underlying recurrent symptomatic HSV infection is not fully understood. In this study, we investigate and characterise the immune response of patients with first episode of HSV genital infection and its relation to the frequency of symptomatic recurrences. Methods In this cohort study, clinical and immunological data were collected from 29 patients who were followed 1 year after presenting with a first episode of genital or meningeal HSV infection. They were classified by PCR and serology as those with primary HSV-1, primary HSV-2 and non-primary HSV-2 infection. Results HSV-specific interleukin(Il)-4 and Il-10 responses at first visit were higher in primary infected HSV-2 infected patients experiencing lower numbers of recurrences during subsequent year. Conclusions The median number of recurrences following primary HSV-2 genital infection may partly be predicted by the strength of an early HSV-specific IL-4 and IL-10 response.

The envelope glycoproteins of herpes simplex virus 1 (HSV-1) and HSV-2, with the exception of glycoprotein G, elicit cross-reactive B- and T-cell responses. Human vaccine trials, using the cross-reactive glycoproteins B and D, have shown no protection against genital HSV-2 infection or disease. In this study, the mature form of glycoprotein G (mgG-2) of HSV-2 was used for immunization of mice, either alone or in combination with adjuvant CpG, followed by an intravaginal challenge with a lethal dose of a fully virulent HSV-2 strain. Mice immunized with mgG-2 plus CpG showed low disease scores and a significantly higher survival rate (73%) than mice immunized with mgG-2 alone (20%) or controls (0%). Accordingly, limited numbers of infectious HSV-2 particles were detected in the spinal cord of mice immunized with mgG-2 plus CpG. The observed protection was associated with a gamma interferon (IFN-γ) response by splenic CD4 + T cells upon antigen restimulation in vitro and in vaginal washes 1 day postinfection. The majority of sera collected from mice immunized with mgG-2 plus CpG showed macrophage-mediated antibody-dependent cellular cytotoxicity and antibody-dependent complement-mediated cytolysis, while no neutralization activity was observed. In conclusion, we have shown that immunization with the type-specific mgG-2 protein in combination with CpG could elicit protective immunity against an otherwise lethal vaginal HSV-2 challenge. The mgG-2 protein may therefore constitute a promising HSV-2 vaccine antigen to be considered for future human trials.

Viral envelope proteins mediate interactions with host cells, leading to internalization and intracellular propagation. Envelope proteins are glycosylated and are known to serve important functions in masking host immunity to viral glycoproteins. However, the viral infectious cycle in cells may also lead to aberrant glycosylation that may elicit immunity. Our knowledge of immunity to aberrant viral glycans and glycoproteins is limited, potentially due to technical limitations in identifying immunogenic glycans and glycopeptide epitopes. This work describes three different complementary methods for high-throughput screening and identification of potential immunodominant O -glycopeptide epitopes on viral envelope glycoproteins: (i) on-chip enzymatic glycosylation of scan peptides, (ii) chemical glycopeptide microarray synthesis, and (iii) a one-bead-one-compound random glycopeptide library. We used herpes simplex virus type 2 (HSV-2) as a model system and identified a simple O -glycopeptide pan-epitope, 501 PPA(GalNAc)TAPG 507 , on the mature gG-2 glycoprotein that was broadly recognized by IgG antibodies in HSV-2-infected individuals but not in HSV-1-infected or noninfected individuals. Serum reactivity to the extended sialyl-T glycoform was tolerated, suggesting that self glycans can participate in immune responses. The methods presented provide new insight into viral immunity and new targets for immunodiagnostic and therapeutic measures.

The clinical picture of herpes simplex virus type 2 (HSV-2) infection includes genital blisters and less frequently meningitis, and some individuals suffer from recurrent episodes of these manifestations. We hypothesized that adaptive and/or innate immune functional deficiencies may be a major contributing factor in susceptibility to recurrent HSV-2 meningitis. Ten patients with recurrent HSV-2 meningitis were studied during clinical remission. For comparison, 10 patients with recurrent genital HSV infections as well as 21 HSV-seropositive and 19 HSV-seronegative healthy blood donors were included. HSV-specific T cell blasting and cytokine secretion were evaluated in whole blood cultures. HSV-2-induced NK cell gamma interferon production, dendritic cell Toll-like receptor (TLR) expression, and TLR agonist-induced alpha interferon secretion were analyzed. Patients with recurrent HSV-2 meningitis had elevated T cell blasting and Th1 and Th2 cytokine production in response to HSV antigens compared to those of patients with recurrent genital infections. A somewhat increased NK cell response, increased dendritic cell expression of TLR3 and -9, and increased TLR-induced alpha interferon responses were also noted. Contrary to our expectation, recurrent HSV-2 meningitis patients have increased HSV-specific adaptive and innate immune responses, raising the possibility of immune-mediated pathology in the development of recurrent HSV2 meningitis.

Summary Background Epidemiological studies point to an inverse relationship between microbial exposure and the prevalence of allergic diseases. The underlying mechanism for this observation remains largely unknown, as well as the nature of the microbes involved. Objective To investigate the effects of early infection with human herpesviruses (HHVs) on IgE formation and T-helper type 2 (Th2) development in infants. Methods Serum was collected from children aged 18 months and assessed for IgE to common allergens and IgG to five common herpesviruses. Cord blood plasmacytoid dendritic cells (pDC) were exposed to HHV type 6 in vitro and mixed with allogeneic cord blood CD4+ T cells. Cytokine levels were determined by ELISA and by flow cytometry. Results We found that children seropositive at 18 months of age to HHV type 6 were significantly less often IgE sensitized than seronegative children [odds ratio (OR): 0.08, 95% confidence interval (CI): 0.009–0.68]. HHV type 6 also decreased the production of the Th2-associated cytokines IL-5 and IL-13 by CD4+ T cells when co-cultured with allogeneic cord blood pDC. This was associated with an increased production of IFN-α by pDC exposed to HHV type 6. Conclusion These data indicate that an early childhood infection with HHV type 6 could down-regulate Th2 responses and reduce IgE formation to common allergens in a young child.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen infecting most individuals worldwide. The majority of HSV-1-infected individuals have no clinical symptoms but shed HSV-1 asymptomatically in saliva. Recent phylogenetic analyses of HSV-1 have defined three genetic clades (A–C) and recombinants thereof. These data have all been based on clinical HSV-1 isolates and do not cover genetic variation of asymptomatically shed HSV-1. The primary goal of this study was to investigate such variation. A total of 648 consecutive saliva samples from five HSV-1-infected volunteers was collected. Asymptomatic shedding was detected on 7.6 % of the days from four subjects. The HSV-1 genome loads were quantified with real-time PCR and varied from 1×102to 2.8×106 copies of virus DNA (ml saliva)−1. Phylogenetic network analyses and bootscanning were performed on asymptomatically shed HSV-1. The analyses were based on DNA sequencing of the glycoprotein I gene, and also of the glycoprotein E gene for putative recombinants. For two individuals with clinical HSV-1 infection, the same HSV-1 strain was shed asymptomatically as induced clinical lesions, and sequence analyses revealed that these strains clustered distinctly to clades A and B, respectively. For one of the subjects with no clinical HSV-1 infection, a recombinant strain was identified. The other truly asymptomatic individual shed evolutionarily distinct HSV-1 strains on two occasions. The first strain was classified as a recombinant and the other strain clustered in clade A. High replication rates of different strains in the same person may facilitate the creation of recombinant clinical HSV-1 strains.

Herpes simplex virus type 2 (HSV-2), human papillomaviruses (HPVs), and Chlamydia trachomatis are the most common pathogens causing sexually transmitted infections (STIs). There is limited information about the prevalences of these STIs in Poland. Here, we estimated the occurrence of immunoglobulin G (IgG) antibodies against HSV-2, HPV, and C. trachomatis in 199 blood donors and 110 patients of both genders attending an STI clinic in Katowice in southern Poland. The seroprevalences of HSV-2 were 5% for blood donors and 14% in the STI cohort. The seroprevalences of the five potentially oncogenic HPV types 16, 18, 31, 35, and 51 were 15%, 7%, 5%, 5%, and 17%, respectively, in blood donors and 37%, 8%, 12%, 5%, and 21%, respectively, in the STI cohort. The majority of HPV-infected individuals showed antibodies against more than one type, i.e., had been infected with multiple HPV types. Anti- C. trachomatis IgG antibodies were detected in 6% of blood donors and 13% of individuals attending the STI clinic. The relatively high prevalence of HPV-51 may have implications for future vaccine programs, as the newly introduced HPV vaccines are based on the potentially oncogenic HPV types 16 and 18.

Glycoprotein I (gI) of herpes simplex virus type 1 (HSV-1) contains a tandem repeat (TR) region including the amino acids serine and threonine, residues that can be utilized forO-glycosylation. The length of this TR region was determined for 82 clinical HSV-1 isolates and the results revealed a polymorphic distribution of two to six or eight repeated blocks with a majority harbouring between two and four repeats. Assessment of theO-glycosylation capacity of an acceptor peptide (STPSTTTSTPSTTT), representing two of the gI blocks, showed that the peptide was a universal substrate forO-glycosylation not only for the two most commonly expressedN-acetyl-d-galactosamine (GalNAc)-T1 and -T2 transferases, but also for the GalNAc-T3, -T4 and -T11 transferases. Immunoblotting of virus-infected cells showed that gI was exclusivelyO-glycosylated with GalNAc monosaccharides (Tn antigen). A polymorphic mucin region has not been described previously for HSV-1 and is a unique finding, as repeated blocks within gI homologues are lacking in other alphaherpesviruses.

Herpes simplex virus (HSV) can enter the central nervous system and cause encephalitis (HSV-1) or meningitis (HSV-2). Microglia, the immunocompetent cells of the central nervous system, are potentially able to detect viral infections. Microglia have been shown to express the glutamate transporter GLT-1 during pathological events, leading to increased microglial glutamate uptake and glutathione synthesis. This study aims to address the role of GLT-1 and glutathione, a major antioxidant with antiviral properties, during HSV infections. Using neuron-enriched mixed primary cultures from rat, it was found that microglia have higher resistance to HSV infections than neurons or astrocytes after 24 h incubation with HSV. Purified microglia in culture were used to further address this. It was found that microglia were able to detect HSV and responded by releasing tumor necrosis factor-alpha (TNF-alpha) and upregulating GLT-1 after 24 h incubation with 1 PFU/cell HSV-1 or HSV-2. Furthermore, the microglial glutathione levels were not significantly diminished after 24 h. Inhibition of the microglial glutathione synthesis with 200 microM buthionine sulfoximide (BSO) led to significantly more infected cells after 24 h incubation with 1 PFU/cell HSV-1 or HSV-2. These data indicate that the higher resistance in microglia against HSV infections may be due to the expression of GLT-1, which can maintain the glutathione levels and provide a mechanism for microglial self-defense against HSV.

Recently, three distinct genotypes of clinical herpes simplex virus type 1 (HSV-1) isolates were identified based on DNA sequence information and phylogenetic analysis of clinical isolates and laboratory strains. We utilized single-nucleotide polymorphism within the genes coding for glycoproteins G and I for rapid genotype classification by PCR and restriction enzyme cleavage. The method is suitable for high-scale genotyping of clinical HSV-1 isolates and for the detection of recombinants.

Herpes simplex virus type 2 (HSV-2) is a common sexually transmitted infection in sub-Saharan Africa. Glycoprotein G (gG) of HSV-2 elicits a type-specific antibody response and is widely used for serodiagnosis. gG is cleaved into a secreted portion (sgG-2) and a highly O-glycosylated mature portion (mgG-2). The performances of these two native immunosorbent purified antigens were compared in an enzyme-linked immunosorbent assay (ELISA) format with a commercially available assay (FOCUS2) using sera from blood donors ( n = 194) and individuals ( n = 198) with genital ulcer disease (GUD) from Tanzania. Discordant results were resolved by Western blotting. The HSV-2 seroprevalence for blood donors was estimated as 42%, and that for the GUD cohort was estimated as 78%. The prevalence increased significantly with age for both cohorts and was higher among human immunodeficiency virus (HIV)-positive individuals than among HIV-negative subjects. In the GUD cohort with a high HSV-2 prevalence, all three assays showed statistically similar performances, with sensitivities between 97% and 99% and specificities in the range of 86% to 91%. In contrast, among blood donors with a lower seroprevalence, the mgG-2-based ELISA presented significantly higher specificity (97%) than the sgG-2 ELISA (89%) and FOCUS2 (74%). Overall, the mgG-2 ELISA gave a high performance, with negative and positive predictive values of 96% for blood donors and a negative predictive value of 95% and a positive predictive value of 97% for the GUD cohort. We conclude that native purified mgG-2 showed the highest accuracy for detection of HSV-2 in patient sera from Tanzania and is therefore suitable for seroprevalence studies as well as in clinical settings.

T-cell recognition of the secreted and membrane-bound portions of the herpes simplex virus type 2 (HSV-2) glycoprotein G (sgG-2 and mgG-2, respectively) was compared in symptomatic and asymptomatic HSV-2-infected individuals and in HSV-2-seronegative controls and the responses with HSV-1 glycoproteins C and E (gC-1 and gE-1) were compared. CD4+T cells from HSV-2-infected individuals specifically recognized both sgG-2 and mgG-2, whereas HSV-1-infected and HSV-seronegative controls did not respond to these glycoproteins. The responses to gC-1 and gE-1, on the other hand, were not type specific, as blood mononuclear cells from both HSV-1- and HSV-2-infected individuals respondedin vitro. There was an association between the status of the infection (symptomatic versus asymptomatic) and the CD4+T-cell responsiveness. Symptomatic HSV-2-seropositive individuals responded with significantly lower Th1 cytokine production to sgG-2 and mgG-2 than did asymptomatic HSV-2-infected carriers, especially within the HSV-1-negative cohort. No differences in T-cell proliferation were observed between asymptomatic and symptomatic individuals. The results have implications for studies of HSV-2-specific CD4+T-cell reactivity in general and for analysis of immunological differences between asymptomatic and symptomatic individuals in particular.

Adaptation of some viruses to replication in cultured cells selects variants that due to alterations in the viral attachment proteins convert to using heparan sulfate (HS) as initial receptor. We report that the nucleotide sequence of herpes simplex virus type 1 (HSV-1) glycoprotein C (gC), a principal attachment component of the virus, remained unchanged during adaptation of wild-type strains to cultured cells. Likewise, amino acid residues critical for binding of gC to HS were conserved in viral strains that replicated in vivo in different human tissues. Moreover wild-type HSV-1 strains derived directly from clinical specimens were, similar to their cell culture propagated progeny viruses and common laboratory strains, sensitive to heparin and demonstrated impairment in their ability to infect HS/chondroitin sulfate deficient cells. These results demonstrate that the HS-binding ability is a feature of wild-type strains of HSV-1.

Glycoprotein G-2 (gG-2) of herpes simplex virus type 2 (HSV-2) is cleaved to a secreted amino-terminal portion (sgG-2) and to a cell-associated carboxy-terminal portion which is further O-glycosylated to constitute the mature gG-2 (mgG-2). In contrast to mgG-2, which is known to elicit a type-specific antibody response in the human host, information on the immunogenic properties of sgG-2 is lacking. Here the sgG-2 protein was purified on a heparin column and used for production of monoclonal antibodies (mAbs). Four anti-sgG-2 mAbs were mapped using a Pepscan technique and identified linear epitopes which localized to the carboxy-terminal part of the protein. One additional anti-sgG-2 mAb, recognizing a non-linear epitope, was reactive to three discrete peptide stretches where the most carboxy-terminally located stretch was constituted by the amino acids 320RRAL323. Although sgG-2 is rapidly secreted into the cell-culture medium after infection, the anti-sgG-2 mAbs identified substantial amounts of sgG-2 in the cytoplasm of HSV-2-infected cells. All of the anti-sgG-2 mAbs were HSV-2 specific showing no cross-reactivity to HSV-1 antigen or to HSV-1-infected cells. Similarly, sera from 50 HSV-2 isolation positive patients were all reactive to sgG-2 in an enzyme immunoassay whilst no reactivity was seen in 25 sera from HSV-1 isolation positive patients or in 25 serum samples from HSV-negative patients suggesting that sgG-2 is a novel antigen potentially suitable for type-discriminating serodiagnosis.

In this study we describe that six rat models (SD, WIST, LEW, BN, F344 and DA) are susceptible to intravaginal herpes simplex virus-2 (HSV-2) infection after pre-treatment with progesterone. At a virus dose of 5 × 10(6) PFU of HSV-2, all rat models were infected presenting anti-HSV-2 antibodies, infectious virus in vaginal washes, and HSV-2 DNA genome copies in lumbosacral dorsal root ganglia and the spinal cord. Most of the LEW, BN, F344, and DA rats succumbed in systemic progressive symptoms at day 8-14 post infection, but presented no or mild genital inflammation while SD and WIST rats were mostly infected asymptomatically. Infected SD rats did not reactivate HSV-2 spontaneously or after cortisone treatment. In an HSV-2 virus dose reduction study, F344 rats were shown to be most susceptible. We also investigated whether an attenuated HSV-1 strain (KOS321) given intravaginally, could protect from a subsequent HSV-2 infection. All LEW, BN, and F344 rats survived a primary HSV-1 infection and no neuronal infection was established. In BN and F344 rats, anti-HSV-1 antibodies were readily detected while LEW rats were seronegative. In contrast to naïve LEW, BN, and F344 rats where only 3 of 18 animals survived 5 × 10(6) PFU of HSV-2, 23 of 25 previously HSV-1 infected rats survived a challenge with HSV-2. The described models provide a new approach to investigate protective effects of anti-viral microbicides and vaccine candidates, as well as to study asymptomatic primary genital HSV-2 infection.

Replication of herpes simplex virus 1 is coupled to recombination, but the molecular mechanisms underlying this process are poorly characterized. The role of Rad51 and Rad52 recombinases in viral recombination was examined in human fibroblast cells 1BR.3.N (wild type) and in GM16097 with replication defects caused by mutations in DNA ligase I. Intermolecular recombination between viruses, tsS and tsK, harboring genetic markers gave rise to ∼17% recombinants in both cell lines. Knock-down of Rad51 and Rad52 by siRNA reduced production of recombinants to 11% and 5%, respectively, in wild type cells and to 3% and 5%, respectively, in GM16097 cells. The results indicate a specific role for Rad51 and Rad52 in recombination of replicating herpes simplex virus 1 DNA. Mixed infections using clinical isolates with restriction enzyme polymorphisms in the US4 and US7 genes revealed recombination frequencies of 0.7%/kbp in wild type cells and 4%/kbp in GM16097 cells. Finally, tandem repeats in the US7 gene remained stable upon serial passage, indicating a high fidelity of recombination in infected cells.

Glycoprotein G (gG-2) of herpes simplex virus type 2 (HSV-2) is cleaved to a secreted amino-terminal portion and to a cell-associated, heavily O-glycosylated carboxy-terminal portion that constitutes the mature gG-2 (mgG-2). The mgG-2 protein is commonly used as a type-specific antigen in the serodiagnosis of HSV-2 infection. As the amino acid sequence variability of mgG-2 in clinical isolates may affect the performance of such assays, the gG-2 gene was sequenced from 15 clinical HSV-2 isolates. Few mutations were identified, and these were mostly localized outside the epitope regions described earlier. Five isolates were identical to different laboratory strains, indicating that the gG-2 gene is highly conserved over time. In the search for HSV-2 isolates harboring mutations within the immunodominant region of mgG-2, a pool of 2,400 clinical HSV-2 isolates was tested for reactivity with two anti-mgG-2 monoclonal antibodies (MAbs). Ten MAb escape HSV-2 mutants, which all harbored structurally restricted single- or dual-point mutations within the respective epitopes explaining the loss of binding, were identified. Sera from corresponding patients were reactive to mgG-2, as well as to a peptide representing the immunodominant region, suggesting that the point mutations detected did not diminish seroreactivity to mgG-2. The conservation of the gG-2 gene reported here further supports the use of mgG-2 as a type-specific antigen in the diagnosis of HSV-2 infections.

Cell surface heparan sulfate (HS) serves as an initial receptor for many different viruses, including herpes simplex virus types 1 and 2 (HSV-1 and 2, respectively). Glycoproteins C and B (gC and gB) are the major components of the viral envelope that mediate binding to HS. In this study, purified gB and gC homologous proteins as well as purified HSV-1 and HSV-2 virions were compared for the ability to bind isolated HS receptor molecules. HSV-1 gC and HSV-2 gC bound comparable amounts of HS. Similarly, HSV-1 gB and its HSV-2 counterpart showed no difference in the HS-binding capabilities. Despite the similar HS-binding potentials of gB and gC homologs, HSV-1 virions bound more HS than HSV-2 particles. Purified gC and gB proteins differed with respect to sensitivity of their interaction with HS to increased concentrations of sodium chloride in the order gB-2 > gB-1 > gC-1 > gC-2. The corresponding pattern for binding of whole HSV virions to cells in the presence of increased ionic strength of the medium was HSV-2 gC-neg1 > HSV-1 gC − 39 > HSV-1 KOS 321 > HSV-2 333. These results relate the HS-binding activities of individual glycoproteins with the cell-binding abilities of whole virus particles. In addition, these data suggest a greater contribution of electrostatic forces for binding of gB proteins and gC-negative mutants compared with binding of gC homologs and wild-type HSV strains. Binding of wild-type HSV-2 virions was the least sensitive to increased ionic strength of the medium, suggesting that the less extensive binding of HS molecules by HSV-2 than by HSV-1 can be compensated for by a relatively weak contribution of electrostatic forces to the binding. Furthermore, gB and gC homologs exhibited different patterns of sensitivity of binding to cells to inhibition with selectively N-, 2-O-, and 6-O-desulfated heparin compounds. The O-sulfate groups of heparin were found to be more important for interaction with gB-1 than gB-2. These results indicate that HSV-1 and HSV-2 differ in their interaction with HS.

Serological diagnosis of herpes simplex virus (HSV) infections requires assays based on antigens that expose type-specific determinants. This study was designed to outline the B-cell epitopes of the type-specific glycoprotein G-1 (gG-1) of HSV type 1 (HSV-1), by investigating the reactivity of human anti-gG-1 antibodies, purified from 21 HSV-1-isolation-proven patient sera, to cellulose-bound synthetic peptides spanning the entire gG-1 sequence. The epitope mapping demonstrated that these antibodies bound preferentially to antigenic determinants that localized to regions with a high degree of amino acid similarity to the corresponding glycoprotein in HSV-2, gG-2. In spite of this, the purified anti-gG-1 antibodies were found to be non-reactive to native gG-2 antigen, as well as to overlapping gG-2 peptides, thus supporting the role of gG-1 as a prototype HSV-1 type-specific antigen. One immunodominant region, delimited by amino acids 112-127, reacted with all purified anti-gG-1 antibodies and may be of interest for the further development of a peptide-based HSV-1 type-specific seroassay.

Glycoprotein G (gG) of herpes simplex virus type 1 (HSV-1) has been used as a prototype antigen for HSV-1 type-specific serodiagnosis, but data on the sequence variability of the gene coding for this protein in wild-type strains are lacking. In this study, direct DNA sequencing of the gG-1 genes from PCR products was performed with clinical HSV-1 isolates from 11 subjects as well as with strains Syn 17 + , F, and KOS 321. The reference strains Syn 17 + and F showed a high degree of conservation, while KOS 321 carried 13 missense mutations and, in addition, 12 silent mutations. Three clinical isolates showed mutations leading to amino acid alterations: one had a mutation of K 122 to N, which is a gG-1–to–gG-2 alteration; another contained all mutations which were observed in KOS 321 except two silent mutations; and the third isolate carried five missense mutations. Two clinical isolates as well as strain KOS 321 showed a mutation (F 111 →V) within the epitope of a gG-1-reactive monoclonal antibody (MAb). When all viruses were tested for reactivity with the anti-gG-1 MAb, the three strains with the F 111 →V mutation were found to be unreactive. Furthermore, gG-1 antibodies purified from sera from the two patients carrying strains mutated in this epitope were less reactive when they were tested by an HSV-1-infected-cell assay. Therefore, our finding that the sequence variability of the gG-1 gene also affects B-cell epitope regions of this protein in clinical isolates may have consequences for the use of this protein as a type-specific antigen for serodiagnosis.