Persistence of Virus-Specific Antibody after Depletion of Memory B Cells.

Humoral immunity is a major component of the adaptive immune response against viruses and other pathogens with pathogen-specific antibody acting as the first line of defense against infection. Virus-specific antibody levels are maintained by continual secretion of antibody by plasma cells residing in the bone marrow. This raises the important question of how the virus-specific plasma cell population is stably maintained and whether memory B cells are required to replenish plasma cells, balancing their loss arising from their intrinsic death rate. In this study, we examined the longevity of virus-specific antibody responses in the serum of mice following acute viral infection with three different viruses: lymphocytic choriomeningitis virus (LCMV), influenza virus, and vesicular stomatitis virus (VSV). To investigate the contribution of memory B cells to the maintenance of virus-specific antibody levels, we employed human CD20 transgenic mice, which allow for the efficient depletion of B cells with rituximab, a human CD20-specific monoclonal antibody. Mice that had resolved an acute infection with LCMV, influenza virus, or VSV were treated with rituximab starting at 2 months after infection, and the treatment was continued for up to a year postinfection. This treatment regimen with rituximab resulted in efficient depletion of B cells (>95%), with virus-specific memory B cells being undetectable. There was an early transient drop in the antibody levels after rituximab treatment followed by a plateauing of the curve with virus-specific antibody levels remaining relatively stable (half-life of 372 days) for up to a year after infection in the absence of memory B cells. The number of virus-specific plasma cells in the bone marrow were consistent with the changes seen in serum antibody levels. Overall, our data show that virus-specific plasma cells in the bone marrow are intrinsically long-lived and can maintain serum antibody titers for extended periods of time without requiring significant replenishment from memory B cells. These results provide insight into plasma cell longevity and have implications for B cell depletion regimens in cancer and autoimmune patients in the context of vaccination in general and especially for COVID-19 vaccines. IMPORTANCE Following vaccination or primary virus infection, virus-specific antibodies provide the first line of defense against reinfection. Plasma cells residing in the bone marrow constitutively secrete antibodies, are long-lived, and can thus maintain serum antibody levels over extended periods of time in the absence of antigen. Our data, in the murine model system, show that virus-specific plasma cells are intrinsically long-lived but that some reseeding by memory B cells might occur. Our findings demonstrate that, due to the longevity of plasma cells, virus-specific antibody levels remain relatively stable in the absence of memory B cells and have implications for vaccination.

The immunogenicity and reactogenicity of four COVID-19 booster vaccinations against SARS-CoV-2 variants following CoronaVac or ChAdOx1 nCoV-19 primary series.

BACKGROUND: The appropriate COVID-19 booster vaccine following inactivated or adenoviral vector COVID-19 vaccination is unclear. OBJECTIVE: To investigate the immunogenicity of four COVID-19 booster vaccines. METHODS: We prospectively enrolled healthy adults who received a two-dose CoronaVac or ChAdOx1 8-12 weeks earlier and allocated them to receive one of the following booster vaccine: inactivated (BBIBP-CorV), ChAdOx1 or mRNA (BNT162b2 at full [30 µg] and half [15 µg] dose) vaccines. We determined the reactogenicity and the humoral (anti-receptor binding domain IgG (anti-RBD-IgG), neutralizing antibodies (nAb) against Delta, Beta and Omicron variants) and cellular immunity measuring by interferon gamma (IFN-γ) responses post-booster. AR patients. RESULTS: Among the 352 participants (179 CoronaVac and 173 ChAdOx1 participants), 285 (81%) were female, and median age was 39 (IQR: 31-47) years. Two weeks post-booster, both 30 µg- and 15 µg- BNT162b2 induced the highest anti-RBD IgG concentration (BAU/mL); Coronavac-prime: 30 µg-BNT162b2, 5152.2 (95%CI 4491.7-5909.8); 15 µg-BNT162b2, 3981.1 (3397.2-4665.4); ChAdOx1, 1358.0 (1141.8-1615.1); BBIBP-CorV, 154.6 (92.11-259.47); ChAdOx1-prime: 30 µg-BNT162b2, 2363.8 (2005.6-2786.1; 15 µg-BNT162b2, 1961.9 (1624.6-2369.1); ChAdOx1, 246.4 (199.6-304.2); BBIBP-CorV, 128.1 (93.5-175.4). Similarly, both 30 µg- and 15 µg- BNT162b2 boosting induced the highest nAb titers against Beta, Delta and Omicron BA.1 variants and highest T-cell response at 2 weeks after boosting. While all BNT162b2 or heterologous ChAdOx1-boosted participants had nAb against Omicron, these were < 50% for BBIBP-CorV and 75% for homologous ChAdOx1-boosted participants. There was significant decrease in nAb ( > 4-fold) at 16-20 weeks post booster for all groups. CONCLUSIONS: Heterologous boosting with BNT162b2 following CoronaVac or ChAdOx1 primary series is most immunogenic. Additional studies are needed to verify the clinical efficacy and persistence of immunity following half-dose BNT162b2.

Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus of significant public health concern. ZIKV shares a high degree of sequence and structural homology compared with other flaviviruses, including dengue virus (DENV), resulting in immunological cross-reactivity. Improving our current understanding of the extent and characteristics of this immunological cross-reactivity is important, as ZIKV is presently circulating in areas that are highly endemic for dengue. To assess the magnitude and functional quality of cross-reactive immune responses between these closely related viruses, we tested acute and convalescent sera from nine Thai patients with PCR-confirmed DENV infection against ZIKV. All of the sera tested were cross-reactive with ZIKV, both in binding and in neutralization. To deconstruct the observed serum cross-reactivity in depth, we also characterized a panel of DENV-specific plasmablast-derived monoclonal antibodies (mAbs) for activity against ZIKV. Nearly half of the 47 DENV-reactive mAbs studied bound to both whole ZIKV virion and ZIKV lysate, of which a subset also neutralized ZIKV. In addition, both sera and mAbs from the dengue-infected patients enhanced ZIKV infection of Fc gamma receptor (FcγR)-bearing cells in vitro. Taken together, these findings suggest that preexisting immunity to DENV may impact protective immune responses against ZIKV. In addition, the extensive cross-reactivity may have implications for ZIKV virulence and disease severity in DENV-experienced populations.

Characterization of Human CD8 T Cell Responses in Dengue Virus-Infected Patients from India.

Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR- CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue. IMPORTANCE: Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects.

Murine gammaherpesvirus 68 encodes a second PML-modifying protein.

The ORF75c tegument protein of murine gammaherpesvirus 68 (MHV68) promotes the degradation of the antiviral promyelocytic leukemia (PML) protein. Surprisingly, MHV68 expressing a degradation-deficient ORF75c replicated in cell culture and in mice similar to the wild-type virus. However, in cells infected with this mutant virus, PML formed novel track-like structures that are induced by ORF61, the viral ribonucleotide reductase large subunit. These findings may explain why ORF75c mutant viruses unable to degrade PML had no demonstrable phenotype after infection.

Promyelocytic leukemia protein modulates establishment and maintenance of latent gammaherpesvirus infection in peritoneal cells.

Promyelocytic leukemia protein (PML) is an essential organizer of PML nuclear bodies (NBs), which carry out a variety of activities, including antiviral functions. Herpesviruses from all subfamilies encode proteins that counteract PML NB-mediated antiviral defenses by multiple mechanisms. However, because of the species specificity of herpesviruses, only a limited number of in vivo studies have been undertaken to investigate the effect of PML or PML NBs on herpesvirus infection. To address this central issue in herpesvirus biology, we studied the course of infection in wild-type and PML⁻/⁻ mice using murine gammaherpesvirus 68 (MHV68), which encodes a tegument protein that induces PML degradation. While acute infection in PML⁻/⁻ mice progressed similarly to that in wild-type mice, the lytic reactivation frequency was higher in peritoneal exudate cells, due to both an increase of MHV68 genome-positive cells and greater reactivation efficiency. We also detected a higher frequency of persistent infection in PML⁻/⁻ peritoneal cells. These findings suggest that the PML protein can repress the establishment or maintenance of gammaherpesvirus latency in vivo. Further use of the PML⁻/⁻ mouse model should aid in dissecting the molecular mechanisms that underlie the role of PML in gammaherpesvirus latency and may yield clues for how PML modulates herpesvirus latency in general.

Comparison of safety and immunogenicity of CoronaVac and ChAdOx1 against the SARS-CoV-2 circulating variants of concern (Alpha, Delta, Beta) in Thai healthcare workers.

Background: Inactivated vaccine (CoronaVac) and chimpanzee adenovirus-vector vaccine (ChAdOx1) have been widely used in resource-limited settings. However, the information on the reactogenicity and immunogenicity of these two vaccines in the same setting are limited. Methods: Healthy health care workers (HCWs) aged 18 years or older were randomly assigned to receive either two doses of CoronaVac at 4 weeks interval or two doses of ChAdOx1 at 10 weeks interval. Self-reported adverse events (AEs) were collected for 7 days following each vaccination. Immunogenicity was determined by IgG antibodies levels against receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S1 subunit) and the 50% plaque reduction neutralization titers against various strains. Results: Of the 360 HCWs, 180 in each vaccine group, the median (interquartile range: IQR) age was 35 (29-44) years old and 84.2% were female. Participants who received ChAdOx1 reported higher frequency of AEs than those received CoronaVac after both the first dose (84.4% vs. 66.1%, P < 0.001) and second dose (75.6% vs. 60.6%, P = 0.002), with more AEs in those younger than 30 years of age for both vaccines. The seroconversion rates were 75.6% and 100% following the first dose of CoronaVac and ChAdOx1, respectively. All participants were seropositive at 2 weeks after the second dose. The anti-SARS-CoV-2 RBD IgG levels induced by CoronaVac was lower than ChAdOX1 with geometric means of 164.4 and 278.5 BAU/mL, respectively (P = 0.0066). Both vaccines induced similar levels of neutralizing antibodies against the Wuhan strain, with the titers of 337.4 and 331.2; however, CoronaVac induced significantly lower GMT against Alpha (23.1 vs. 92.5), Delta (21.2 vs. 69.7), and Beta (10.2 vs. 43.6) variants, respectively. Conclusion: CoronaVac induces lower measurable antibodies against circulating variants but with lower frequency of AEs than ChAdOx1. An earlier boosting to prevent breakthrough infections may be needed.

Murine gammaherpesvirus 68 open reading frame 75c tegument protein induces the degradation of PML and is essential for production of infectious virus.

Promyelocytic Leukemia nuclear body (PML NB) proteins mediate an intrinsic cellular host defense response against virus infections. Herpesviruses express proteins that modulate PML or PML-associated proteins by a variety of strategies, including degradation of PML or relocalization of PML NB proteins. The consequences of PML-herpesvirus interactions during infection in vivo have yet to be investigated in detail, largely because of the species-specific tropism of many human herpesviruses. Murine gammaherpesvirus 68 (gammaHV68) is emerging as a suitable model to study basic biological questions of virus-host interactions because it naturally infects mice. Therefore, we sought to determine whether gammaHV68 targets PML NBs as part of its natural life cycle. We found that gammaHV68 induces PML degradation through a proteasome-dependent mechanism and that loss of PML results in more robust virus replication in mouse fibroblasts. Surprisingly, gammaHV68-mediated PML degradation was mediated by the virion tegument protein ORF75c, which shares homology with the cellular formylglycinamide ribotide amidotransferase enzyme. In addition, we show that ORF75c is essential for production of infectious virus. ORF75 homologs are conserved in all rhadinoviruses but so far have no assigned functions. Our studies shed light on a potential role for this unusual protein in rhadinovirus biology and suggest that gammaHV68 will be a useful model for investigation of PML-herpesvirus interactions in vivo.

IgG antibodies to dengue enhanced for FcγRIIIA binding determine disease severity.

Dengue virus (DENV) infection in the presence of reactive, non-neutralizing immunoglobulin G (IgG) (RNNIg) is the greatest risk factor for dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Progression to DHF/DSS is attributed to antibody-dependent enhancement (ADE); however, because only a fraction of infections occurring in the presence of RNNIg advance to DHF/DSS, the presence of RNNIg alone cannot account for disease severity. We discovered that DHF/DSS patients respond to infection by producing IgGs with enhanced affinity for the activating Fc receptor FcγRIIIA due to afucosylated Fc glycans and IgG1 subclass. RNNIg enriched for afucosylated IgG1 triggered platelet reduction in vivo and was a significant risk factor for thrombocytopenia. Thus, therapeutics and vaccines restricting production of afucosylated, IgG1 RNNIg during infection may prevent ADE of DENV disease.

Murine gammaherpesvirus 68 ORF75c contains ubiquitin E3 ligase activity and requires PML SUMOylation but not other known cellular PML regulators, CK2 and E6AP, to mediate PML degradation.

All gammaherpsviruses encode at least one gene related to the cellular formylglycinamide ribonucleotide amidotransferase (FGARAT) enzyme but their biological roles are relatively unknown. The murine gammaherpesvirus 68 (MHV68) vFGARAT, ORF75c, mediates a proteasome-dependent degradation of the antiviral promyelocytic leukemia (PML) protein by an unknown mechanism, which is addressed in this study. We found that ORF75c interacts weakly with PML and SUMO-modified forms of PML are important for its degradation by ORF75c. ORF75c-mediated PML degradation was not dependent on two known cellular regulators of PML stability, Casein kinase II (CK2) and human papilloma virus E6-associated protein (E6AP). Finally, ORF75c had self-ubiquitination activity in vitro and its expression increased levels of ubiquitinated PML in transfected cells. Taken together, the evidence accumulated in this study provides new insights into the function of a vFGARAT and is consistent with a model in which ORF75c could mediate direct ubiquitination of PML resulting in its degradation by the proteasome.

Compositional bias and size of genomes of human DNA viruses.

Genomes of 144 human DNA viruses were analyzed in the aspect of their compositional asymmetry. DNA viruses were divided into two groups according to their genome sizes. The analysis revealed that the level of guanine and cytosine (GC content) in the coding sequences of small genome DNA viruses was significantly lower than that of large genome DNA viruses. Because small genome viruses replicate their genomes using cellular enzymes, while large genome viruses use their own enzymes for genome replication, the two groups of viruses may be under different mutational bias and/or selection pressure. In these viruses, GC content at the third codon position correlated with GC content at the first and second codon position. However, the relationship in small genome DNA viruses was weaker than that in large genome DNA viruses, suggesting that their genome composition may be more strongly influenced by codon usage preference or restriction on amino acid composition.