Our vaccine candidate for genital herpes includes three immunogens involved in virus entry and immune evasion. HSV-2 glycoprotein C (gC2) is one of the immune evasion molecules that inhibits complement activation by binding C3b, and is the focus of this manuscript. Mice were immunized with 0.25, 0.5, 1, 10, or 30 µg of gC2 lipid nano particle (LNP)-encapsulated nucleoside-modified mRNA and challenged intravaginally with HSV-2. The gC2 mRNA-LNP, even at the lowest dose, was highly protective as a single immunogen. We measured antibody responses to six gC2 epitopes. Both neutralizing and C3b binding epitopes on gC2 were targeted. We passively immunized mice with monoclonal antibodies (mAbs) to gC2 and determined that the mAbs that enhance complement activation by blocking C3b binding were protective, while the mAb that neutralizes the virus, but does not block C3b binding, failed to protect. These results highlight the importance of a vaccine immunogen that induces antibodies that block the ability of gC to inhibit complement activation.

Despite the existence of a highly effective vaccine, measles may appear in some vaccinated individuals after exposure to wild type measles virus. Reduced circulation of the measles virus in the population in low-endemic countries results in an absence of viral challenge and may cause waning of immunity over time after vaccination. Another reason for breakthrough infection is primary vaccine failure due to immunological conditions or defective measles virus vaccine. Exposition to a high dose of measles virus without proper protective respiratory masks may increase the risk of breakthrough measles, as has been reported in some outbreaks where health care workers were exposed. The clinical symptoms in breakthrough measles are often milder than in primary measles. Respiratory symptoms and conjunctivitis are less common than in primary measles. The contagiousness of breakthrough measles is low and in some cases negligible.

The role of glycoprotein G (gG-2) of herpes simplex virus type 2 (HSV-2) in viral pathogenesis remains poorly understood. gG-2 is cleaved into a secreted form (sgG-2) and a membrane-associated form (mgG-2), but the in vivo function of mgG-2 and the contribution of its glycosylation to immune responses have not been defined. Here, we provide a comprehensive characterization of the N- and O-linked glycosylation profile of mgG-2 and investigate its functional relevance for viral spread and vaccine-induced immunity. Using a mouse genital infection model, we show that an mgG-2-deficient HSV-2 mutant replicates in vaginal epithelial cells but is severely impaired in dissemination to dorsal root ganglia and the central nervous system, identifying mgG-2 as a key determinant of neuronal spread in vivo. In parallel, immunization with recombinant mgG-2 elicited strong humoral and Th1-polarized CD4 + T-cell responses and conferred protection against genital HSV-2 challenge. Importantly, glycosylation of mgG-2 was required for optimal immunogenicity and protection, as deglycosylated variants induced reduced CD4 + T-cell responses and allowed increased viral spread to neuronal tissues. Mechanistically, our findings suggest that glycosylation of mgG-2 modulates antigen recognition and shapes adaptive immune responses that limit viral dissemination after vaccination. Together, these results demonstrate that mgG-2 plays a critical role in HSV-2 pathogenesis and provide a strong rationale for targeting glycosylated mgG-2 in the development of both prophylactic and therapeutic vaccines against HSV-2.

Epstein-Barr virus (EBV) is a critical risk factor for multiple sclerosis (MS), but the pathogenic mechanisms remain elusive. To clarify its role in MS, we examined the sequence of EBV-related antibody responses over a broad period before the clinical onset of MS, from early childhood to middle age. A nested case-control study was performed by linking Swedish MS registries with biobanks to identify pre-symptomatic samples from individuals who later developed MS and matched controls. Leveraging these samples, we analysed the immune responses to EBV in and before the prodromal phase of MS. Antibodies against EBV (viral capsid antigen, VCA; EBV nuclear antigen 1, EBNA1; early antigen-diffuse, EA-D) and the putative autoantibody target Anoctamin 2 (ANO2) were quantified by immunoassay. Serum neurofilament light chain (S-NfL), a marker of axonal injury, was quantified by single-molecule array. Ratios of these markers were calculated within each matched case-control set, plotted against time to the clinical onset of MS, and analysed with Loess regression to visualise the temporal order of events on group level. Samples from 981 cases and 1278 controls were included. Median age at blood sampling was 22 years and 32% of the participants were children or adolescents. The median time from the blood sample to the clinical onset of MS was 9 years. On the group level, we observed the following sequence of events: Seroreactivity against lytic EBV antigens (VCA-IgG, VCA-IgM, EA-D) - characterizing primary EBV infection and reactivation - were significantly higher in cases than in controls more than 20 years before MS onset and onwards. Increased seroreactivity against the latent antigen EBNA1 was observed 15 years before onset, followed by increased ANO2 seroreactivity from 9 years and onwards. Finally, a significant elevation of S-NfL was observed from 7 years prior to MS onset. These findings demonstrate a more extensive lytic EBV infection in the decades before MS onset, consistent with the hypothesis that EBV is a driver of MS development. The sequence of increasing seroreactivity against EBNA1 and ANO2 was closely followed by biochemical signs of neuroaxonal injury, suggesting epitope spreading from EBNA1 before the subclinical onset of neuroaxonal damage. This sequence of events supports the hypothesis that EBV infection promotes autoreactive immune cells contributing to MS pathogenesis.

Introduction: HSV-2 glycoproteins C, D, and E (gC2/gD2/gE2) are immunogens included in an experimental HSV-2 vaccine. We evaluated whether these antigens serve as targets for antibody-dependent cellular cytotoxicity (ADCC). Methods: We transiently transfected HEK cells with gC2/gD2/gE2 DNA, added HSV-2 seropositive human convalescent sera (HCS), and measured surface CD107a expression on human NK cells by flow cytometry. Results: We demonstrated that antibodies to gC2/gD2/gE2 mediate ADCC. HSV-2 gE and gI form a complex that binds IgG Fc. We next determined whether gE2/gI2 inhibits ADCC, a crucial function mediated by the IgG Fc, by comparing ADCC titers when HCS were added to cells transfected with gD2, gI2, and a gE2 mutant (gE2(MUT)) unable to bind IgG Fc or gD2, gI2, and gE2 wild-type (gE2(WT)). ADCC titers increased by 6.5-fold when cells were transfected with the gE2(MUT) versus gE2(WT) (P=0.01). We then spiked HCS with a gE2 mAb that blocks IgG Fc binding, or with a gE2 mAb that does not block IgG Fc binding. The blocking mAb significantly increased titers (P<0.0001), whereas a non-blocking gE2 mAb had no effect. Discussion: We conclude that antibodies to gC2/gD2/gE2 are targets of ADCC, that gE2/gI2 inhibits ADCC, and that an mAb that targets the gE2 IgG Fc binding domain can prevent this inhibition.

The function of glycoprotein G (gG-2) of herpes simplex virus 2 (HSV-2) during genital infection is unknown. gG-2 is cleaved into a secreted variant (sgG-2) and a membrane associated variant (mgG-2). This work delineates the glycan profile of mgG-2, and demonstrates that mgG-2 is strongly immunogenic, eliciting glycan dependent humoral and Th1-polarized CD4+ T cell responses in a mouse vaccination model. The N- and O-glycosylation of mgG-2 is important to achieve full protection against HSV-2 infection as immunization with deglycosylated mgG-2 resulted in poor CD4+ T cell activation and viral spread to dorsal root ganglia and spinal cord. Furthermore, an mgG-2 negative HSV-2 mutant virus failed to spread to the dorsal root ganglia and the central nervous system of genitally infected mice, despite viral replication in vaginal cells. Our data demonstrate that mgG-2 is important for HSV-2 propagation and that adaptive immune responses targeting the glycosylated protein can prevent neuronal infection, identifying mgG-2 as a promising vaccine candidate.

BACKGROUND AND OBJECTIVES:Biomarkers for predicting disease severity and outcome in Guillain-Barré syndrome (GBS) are scarce. We aimed to determine if brain-derived tau in serum (sBD-tau) and cerebrospinal fluid (CSF BD-tau) are associated with long-term outcome and disease severity in GBS. METHODS:In this retrospective study of 100 GBS patients, we measured sBD-tau and CSF BD-tau at diagnosis. Outcome was defined as GBS disability scale (GBSDS) > 2 and overall neuropathy limitation scale (ONLS) at 12 months, disease severity as respiratory support and ONLS at nadir. BD-tau levels were compared between groups and correlated with ONLS scores. Regression analyses and receiver operator characteristic curve analyses were performed for GBSDS > 2 at 12 months. RESULTS:BD-tau levels were higher for GBSDS > 2 at 12 months in serum and CSF. Odds ratio for sBD-tau was 1.9 (95% CI 1.08-3.2, p = 0.03) and for CSF BD-tau was 5.9 (95% CI 1.4-25, p = 0.02). Area under curve for sBD-tau was 0.75 (95% CI 0.57-0.9, p < 0.001) and for CSF BD-tau was 0.78 (95% CI 0.65-0.9, p = 0.001). ONLS at 12 months correlated with sBD-tau (ρ = 0.34 [95% CI 0.12-0.53], p = 0.002) and CSF BD-tau (ρ = 0.33 [95% CI 0.08-0.54], p = 0.01). Statistically significant difference in BD-tau levels was not seen for respiratory support or ONLS at nadir. CONCLUSION:BD-tau at GBS onset is associated with long-term outcome but not disease severity. Because BD-tau is essentially a CNS biomarker, our results suggest that CNS involvement influences recovery.

Background: The knowledge of traditional health practitioners (THPs) on the use of medicinal plant remedies for treatment of human respiratory ailments provides a potential resource for identification of relevant plants and isolation of active ingredients. Purpose: We aimed to identify medicinal plants used by THPs for treatment of respiratory ailments, then screen them in cultured cells for activity against respiratory syncytial virus (RSV), and assess the antiviral potential of identified screening hits. Methods: Forty Rwandese THPs were interviewed to identify medicinal plants used for treatment of respiratory ailments. The indicated plant samples were collected and successively extracted using n-hexane, ethyl acetate, methanol, and water. Extracts were screened for anti-RSV activity and for cytotoxicity in HEp-2 cells. Results: Based on ethnopharmacological survey of THPs, 48 plants used for treating respiratory ailments were identified. Of 203 plant extracts prepared for screening, ten methanolic and four aqueous extracts exhibited anti-RSV activity, manifested by EC50 values ranging from 1.6 to 39.5 µg/ml and selectivity indices (SI, CC50/EC50) of 12–294. Extended studies of an anti-RSV potential of methanolic extract of Senecio maranguensis O. Hoffm. leaves revealed that this preparation targeted the virus fusion (F) protein because extract resistant virus variants carried the double nucleotide substitution at c1104t and a1105g resulting in the T335M amino acid change in the F protein trimer internal cavity. Conclusion: We found that eleven plants, traditionally used against respiratory ailments, exhibited substantial anti-RSV activity in cultured cells, exemplified by the S. maranguensis methanolic extract that affected the activity of RSV F protein.

The phytochemical investigation of the stem bark of Morus mesozygia afforded the fourteen known secondary metabolites moracin N (1), moracin S (2), mulberrofuran L (3), moracin C (4), moracin L (5), moracin M (6), moracin D (7), artopithecin A (8), isobavachalcone (9), morachalcone A (10), 2,2՛,4,4՛-tetrahydroxychalcone (11), 3β-acetoxy-urs-12-en-11-one (12), betulinic acid (13), and 4,4'-diphenylmethane-bis(methyl) carbamate (14). Their structures were elucidated by NMR spectroscopic and mass spectrometric analyzes and their cytotoxicity (CC₅₀ for HEKa, IMR-90, and HPrEC), anti-inflammatory (TNF-α, NF-κB, and NO inhibition), antibacterial (MIC), antitumor (IC₅₀), and antiviral (CPE-based and plaque reduction assays) activities were studied. Moracin D (7) showed potent anti-inflammatory activity towards the release of NF-κB (0.57 < IC50 < 1.21 μM). 3β-Acetoxy-urs-12-en-11-one (12) had potent antibacterial activity towards Bacillus subtilis and Micrococcus luteus, with MIC values of 12.71 and 15.59 μM, respectively. Moracin M (6) had the highest antitumor activity (IC50 = 19.80 μM) against SK-MEL-28 human melanoma cells. Isobavachalcone (9) exhibited activity against Human Rhinovirus 2 (HRV-2; IC50 = 7.01 μM) with a selectivity index (SI) = 9.1 as compared to HeLa cells. None of the compounds exhibited significant antiviral activity against respiratory syncytial virus (RSV) or herpes simplex virus type 2 (HSV-2). Out of the isolated compounds, moracin D (7), isobavachalcone (9) and marsformoxide B (12), may be considered to be promising leads for the development of anti-inflammatory (NF-κB), antiviral (HRV-2), and antibacterial (B. subtilis and M. luteus) agents, respectively.

Epstein–Barr virus is now regarded as the critical risk factor for multiple sclerosis. However, Cytomegalovirus and human herpesvirus 6A have also been associated with altered multiple sclerosis risk, suggesting a multifactorial aetiology. Here, we present the first large-scale study of the association between human herpesvirus 7 and the risk of developing multiple sclerosis. A nested case-control study was performed by crosslinking Swedish registries and biobanks, identifying blood samples from 981 cases who later developed multiple sclerosis and 1278 matched controls. Serological testing was performed with a multiplex immunoassay. The association between viral serostatus and the risk of developing multiple sclerosis was analysed with conditional logistic regression, calculating an odds ratio with 95% confidence interval. Interactions between antibodies against human herpesvirus 7 and the Epstein–Barr virus nuclear antigen 1 regarding multiple sclerosis risk were analysed on the additive scale. Serological evidence of human herpesvirus 7 infection was associated with a higher risk of developing multiple sclerosis: odds ratio = 2.2 (95% confidence interval = 1.8–2.7), P < 0.001. The results remained similar when adjusting for cytomegalovirus, Epstein–Barr virus and human herpesvirus 6A serostatus. Synergistic interactions between human herpesvirus 7 and Epstein–Barr virus nuclear antigen 1 seroreactivity were observed: attributable proportion due to interaction = 0.51 (95% confidence interval = 0.34–0.68). These results suggest that human herpesvirus 7 could be a contributing factor in multiple sclerosis aetiology.

BACKGROUND:Tick-borne encephalitis (TBE) is a neurological disease caused by the tick-borne encephalitis virus (TBEV). Despite available vaccines, breakthrough infections occur, some fatal. OBJECTIVES:As no antiviral therapy for TBE is currently approved, this study evaluated the in vitro activity of already licenced remdesivir (RDV) and sofosbuvir (SOF) for possible drug repurposing against TBEV. METHODS:TBEV was cultured in A549 cells, and the inhibitory effects of RDV (GS-5734), its parent nucleotide GS-441524, and SOF (GS-7977) were assessed. RESULTS:After 78 h, RDV demonstrated significantly lower EC50 values than SOF (0.14 vs. 11 µM) based on TBEV RNA levels measured by RT-qPCR. RDV also had a lower mean EC50 (0.55 µM) compared to GS-441524 and SOF (>8.9 and 13.1 µM, respectively) using crystal violet staining after 5 days. After 11 passages of TBEV in the presence of RDV, emergence of virus with a higher EC50 (1.32 vs. 0.55 µM) was detected with two mutations (L3122F and Y3278F) in NS5, the viral RNA-dependent RNA polymerase (RdRp), and one substitution in envelope (E) protein (E402G). Similarly, SOF resistance appeared after 20 passages, increasing EC50 values (35.5 vs. 10 µM). CONCLUSION:RDV exhibits potent in vitro antiviral activity against TBEV via specific targeting of the viral RdRp as confirmed by the emergence of resistance-associated double NS5 substitutions in vitro in the presence of RDV. While the potential in vivo implications of the observed RDV resistance remain to be determined, these in vitro data support further assessment of RDV for the treatment of TBEV infection.

Herpes zoster (HZ) is a painful disease that mainly affects individuals whose immune system has been weakened because of increasing age (> 50 years) or certain diseases or treatments. We estimated the complete burden of HZ. This population-based register study analysed healthcare data from the VEGA and Digitalis databases of Västra Götaland Region (VGR), Sweden. The VEGA database includes all patients in VGR, covering both hospital and primary care. The Digitalis records prescribed medications. The study population included patients aged ≥ 18 years with at least one registered primary or secondary HZ diagnosis (based on International Classification of Diseases [ICD] codes) between 2005 and 2021. Incidence rates (95 www.clinicaltrials.gov ). What is the context? The varicella-zoster virus (VZV) can reactivate after primary infection and cause herpes zoster or shingles. Shingles is painful and mostly affects people aged > 50 years or those who have weakened immunity due to age, illness or medical treatments. The National Immunization Program (NIP) in Sweden does not currently include vaccination against shingles. We evaluated how often individuals from Västra Götaland Region, Sweden, experienced shingles and its complications. What is new? Overall, the number of shingles and PHN cases increased significantly from 2005 to 2021. We found that 14

BackgroundMultiple sclerosis (MS) and presymptomatic axonal injury appear to develop only after an Epstein-Barr virus (EBV) infection. This association remains to be confirmed across a broad preclinical time range, for lytic and latent EBV seroreactivity, and for potential cross-reacting antigens.MethodsWe performed a case–control study with 669 individual serum samples obtained before clinical MS onset, identified through cross-linkage with the Swedish MS register. We assayed antibodies against EBV nuclear antigen 1 (EBNA1), viral capsid antigen p18, glycoprotein 350 (gp350), the potential cross-reacting protein anoctamin 2 (ANO2) and the level of sNfL, a marker of axonal injury.ResultsEBNA1 (latency) seroreactivity increased in the pre-MS group, at 15–20 years before clinical MS onset, followed by gp350 (lytic) seroreactivity (p=0.001–0.009), ANO2 seropositivity appeared shortly after EBNA1-seropositivity in 16.7% of pre-MS cases and 10.0% of controls (p=0.001).With an average lag of almost a decade after EBV, sNfL gradually increased, mainly in the increasing subgroup of seropositive pre-MS cases (p=8.10−5compared with non-MS controls). Seropositive pre-MS cases reached higher sNfL levels than seronegative pre-MS (p=0.038). In the EBNA1-seropositive pre-MS group, ANO2 seropositive cases had 26% higher sNfL level (p=0.0026).ConclusionsSeroreactivity against latent and lytic EBV antigens, and in a subset ANO2, was detectable on average a decade before the appearance of a gradually increasing axonal injury occurring in the last decade before the onset of clinical MS. These findings strengthen the hypothesis of latent EBV involvement in the pathogenesis of MS.

BackgroundIn Sweden, information on seroprevalence of tick-borne encephalitis virus (TBEV) in the population, including vaccination coverage and infection, is scattered. This is largely due to the absence of a national tick-borne encephalitis (TBE) vaccination registry, scarcity of previous serological studies and use of serological methods not distinguishing between antibodies induced by vaccination and infection. Furthermore, the number of notified TBE cases in Sweden has continued to increase in recent years despite increased vaccination.AimThe aim was to estimate the TBEV seroprevalence in Sweden.MethodsIn 2018 and 2019, 2,700 serum samples from blood donors in nine Swedish regions were analysed using a serological method that can distinguish antibodies induced by vaccination from antibodies elicited by infection. The regions were chosen to reflect differences in notified TBE incidence.ResultsThe overall seroprevalence varied from 9.7% (95% confidence interval (CI): 6.6-13.6%) to 64.0% (95% CI: 58.3-69.4%) between regions. The proportion of vaccinated individuals ranged from 8.7% (95% CI: 5.8-12.6) to 57.0% (95% CI: 51.2-62.6) and of infected from 1.0% (95% CI: 0.2-3.0) to 7.0% (95% CI: 4.5-10.7). Thus, more than 160,000 and 1,600,000 individuals could have been infected by TBEV and vaccinated against TBE, respectively. The mean manifestation index was 3.1%.ConclusionA difference was observed between low- and high-incidence TBE regions, on the overall TBEV seroprevalence and when separated into vaccinated and infected individuals. The estimated incidence and manifestation index argue that a large proportion of TBEV infections are not diagnosed.

Synergistic interactions between human herpesvirus 6A (HHV-6A) and Epstein-Barr virus (EBV) are hypothesized in the etiopathogenesis of multiple sclerosis (MS). This study investigated if HHV-6A and EBV seroreactivities interact regarding the risk of developing MS. Antibodies against viral antigens were analyzed in biobank samples from 670 individuals who later developed MS and matched controls. Additive interactions were analyzed. A significant interaction between HHV-6A and EBNA-1 seroreactivities was observed in study participants above the median age of 24.9 years (attributable proportion due to interaction = 0.45). This finding supports the hypothesis that HHV-6A and EBV infections interact in MS development. ANN NEUROL 2024;96:302-305.

Three new (1-3) and six known rotenoids (5-10), along with three known isoflavones (11-13), were isolated from the leaves of Millettia oblata ssp. teitensis. A new glycosylated isoflavone (4), four known isoflavones (14-18), and one known chalcone (19) were isolated from the root wood extract of the same plant. The structures were elucidated by NMR and mass spectrometric analyses. The absolute configuration of the chiral compounds was established by a comparison of experimental ECD and VCD data with those calculated for the possible stereoisomers. This is the first report on the use of VCD to assign the absolute configuration of rotenoids. The crude leaves and root wood extracts displayed anti-RSV (human respiratory syncytial virus) activity with IC50 values of 0.7 and 3.4 mu g/mL, respectively. Compounds 6, 8, 10, 11, and 14 showed anti-RSV activity with IC50 values of 0.4-10 mu M, while compound 3 exhibited anti-HRV-2 (human rhinovirus 2) activity with an IC50 of 4.2 mu M. Most of the compounds showed low cytotoxicity for laryngeal carcinoma (HEp-2) cells; however compounds 3, 11, and 14 exhibited low cytotoxicity also in primary lung fibroblasts. This is the first report on rotenoids showing antiviral activity against RSV and HRV viruses.

Recent research indicates that multiple sclerosis is preceded by a prodromal phase with elevated levels of serum neurofilament light chain (sNfL), a marker of axonal injury. The effect of environmental risk factors on the extent of axonal injury during this prodrome is unknown. Human herpesvirus 6A (HHV-6A) is associated with an increased risk of developing multiple sclerosis. The objective of this study was to determine if HHV-6A serostatus is associated with the level of sNfL in the multiple sclerosis prodrome, which would support a causative role of HHV-6A.A nested case-control study was performed by crosslinking multiple sclerosis registries with Swedish biobanks. Individuals with biobank samples collected before the clinical onset of multiple sclerosis were included as cases. Controls without multiple sclerosis were randomly selected, matched for biobank, sex, sampling date and age. Serostatus of HHV-6A and Epstein-Barr virus was analysed with a bead-based multiplex assay. The concentration of sNfL was analysed with single molecule array technology. The association between HHV-6A serology and sNfL was assessed by stratified t-tests and linear regressions, adjusted for Epstein-Barr virus serostatus and sampling age. Within-pair ratios of HHV-6A seroreactivity and sNfL were calculated for each case and its matched control. To assess the temporal relationship between HHV-6A antibodies and sNfL, these ratios were plotted against the time to the clinical onset of multiple sclerosis and compared using locally estimated scatterplot smoothing regressions with 95% confidence intervals (CI).Samples from 519 matched case-control pairs were included. In cases, seropositivity of HHV-6A was significantly associated with the level of sNfL (+11%, 95% CI 0.2-24%, P = 0.045) and most pronounced in the younger half of the cases (+24%, 95% CI 6-45%, P = 0.007). No such associations were observed among the controls. Increasing seroreactivity against HHV-6A was detectable before the rise of sNfL (significant within-pair ratios from 13.6 years versus 6.6 years before the clinical onset of multiple sclerosis).In this study, we describe the association between HHV-6A antibodies and the degree of axonal injury in the multiple sclerosis prodrome. The findings indicate that elevated HHV-6A antibodies both precede and are associated with a higher degree of axonal injury, supporting the hypothesis that HHV-6A infection may contribute to multiple sclerosis development in a proportion of cases. Grut et al. uncover an association between prior infection with human herpesvirus 6A (HHV-6A) and subtle axonal injury-as revealed by serum neurofilament light levels-in younger individuals who later develop multiple sclerosis. They conclude that HHV-6A infection may contribute to multiple sclerosis development in a proportion of cases.

Herpes simplex virus 2 (HSV-2) is a sexually transmitted infection affecting 491 million individuals globally. Consequently, there is a great need for both prophylactic and therapeutic vaccines. Unfortunately, several vaccine clinical trials, primarily employing the glycoprotein D of HSV-2 (gD-2), have failed. The immune protection conferred by human anti-HSV-2 antibodies in genital infection and disease remains elusive. It is well-known that gD-2 elicits cross-reactive neutralizing antibodies, i.e., anti-gD-2 antibodies recognize gD in HSV-1 (gD-1). In contrast, anti-glycoprotein G in HSV-2 (mgG-2) antibodies are exclusively type-specific for HSV-2. In this study, truncated versions of gD-2 and mgG-2 were recombinantly produced in mammalian cells and used for the purification of anti-gD-2 and anti-mgG-2 antibodies from the serum of five HSV-2-infected subjects, creating a pool of purified antibodies. These antibody pools were utilized as standards together with purified mgG-2 and gD-2 antigens in ELISA to quantitatively estimate and compare the levels of cross-reactive anti-gD-1 and anti-gD-2 antibodies, as well as anti-mgG-2 antibodies in sera from HSV-1+2-, HSV-2-, and HSV-1-infected subjects. The median concentration of anti-mgG-2 antibodies was five times lower in HSV-1+2-infected subjects as compared with cross-reactive anti-gD-1 and anti-gD-2 antibodies, and three times lower in HSV-2 infected subjects as compared with anti-gD-2 antibodies. The pool of purified anti-gD-2 antibodies presented neutralization activity at low concentrations, while the pool of purified anti-mgG-2 antibodies did not. Instead, these anti-mgG-2 antibodies mediated antibody-dependent cellular cytotoxicity (ADCC) by human granulocytes, monocytes, and NK-cells, but displayed no complement-dependent cytotoxicity. These findings indicate that antibodies to mgG-2 in HSV-2-infected subjects are present at low concentrations but mediate the killing of infected cells via ADCC rather than by neutralizing free viral particles. We, and others, speculate that Fc-receptor mediated antibody functions such as ADCC following HSV-2 vaccination may serve as a better marker of protection correlate instead of neutralizing activity. In an mgG-2 therapeutic vaccine, our findings of low levels of anti-mgG-2 antibodies in HSV-2-infected subjects may suggest an opportunity to enhance the immune responses against mgG-2. In a prophylactic HSV-2 mgG-2 vaccine, a possible interference in cross-reactive immune responses in already infected HSV-1 subjects can be circumvented.

ABSTRACT Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC 50 of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections.