Transcription of HIV-1 at sites of intact latent provirus integration.

HIV-1 antiretroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses, leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here, we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 100-10,000× less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir, thereby influencing cytopathic effects and proviral immune evasion.

Quantitative trait loci mapping provides insights into the genetic regulation of dendritic cell numbers in mouse tissues.

To explore the influence of genetics on homeostatic regulation of dendritic cell (DC) numbers, we present a screen of DCs and their progenitors in lymphoid and non-lymphoid tissues in Collaborative Cross (CC) and Diversity Outbred (DO) mice. We report 30 and 71 loci with logarithm of the odds (LOD) scores >8.18 and ranging from 6.67 to 8.19, respectively. The analysis reveals the highly polygenic and pleiotropic architecture of this complex trait, including many of the previously identified genetic regulators of DC development and maturation. Two SNPs in genes potentially underlying variation in DC homeostasis, a splice variant in Gramd4 (rs235532740) and a missense variant in Orai3 (rs216659754), are confirmed by gene editing using CRISPR-Cas9. Gramd4 is a central regulator of DC homeostasis that impacts the entire DC lineage, and Orai3 regulates cDC2 numbers in tissues. Overall, the data reveal a large number of candidate genes regulating DC homeostasis in vivo.

Human antibodies in Mexico and Brazil neutralizing tick-borne flaviviruses.

Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV) are spread by mosquitoes and cause human disease and mortality in tropical areas. In contrast, Powassan virus (POWV), which causes severe neurologic illness, is a flavivirus transmitted by ticks in temperate regions of the Northern hemisphere. We find serologic neutralizing activity against POWV in individuals living in Mexico and Brazil. Monoclonal antibodies P002 and P003, which were derived from a resident of Mexico (where POWV is not reported), neutralize POWV lineage I by recognizing an epitope on the virus envelope domain III (EDIII) that is shared with a broad range of tick- and mosquito-borne flaviviruses. Our findings raise the possibility that POWV, or a flavivirus closely related to it, infects humans in the tropics.

Bispecific antibodies with broad neutralization potency against SARS-CoV-2 variants of concern.

The ongoing emergence of SARS-CoV-2 variants of concern (VOCs) that reduce the effectiveness of antibody therapeutics necessitates development of next-generation antibody modalities that are resilient to viral evolution. Here, we characterized N-terminal domain (NTD) and receptor binding domain (RBD)-specific monoclonal antibodies previously isolated from COVID-19 convalescent donors for their activity against emergent SARS-CoV-2 VOCs. Among these, the NTD-specific antibody C1596 displayed the greatest breadth of binding to VOCs, with cryo-EM structural analysis revealing recognition of a distinct NTD epitope outside of the site i antigenic supersite. Given C1596's favorable binding profile, we designed a series of bispecific antibodies (bsAbs) termed CoV2-biRNs, that featured both NTD and RBD specificities. Notably, two of the C1596-inclusive bsAbs, CoV2-biRN5 and CoV2-biRN7, retained potent in vitro neutralization activity against all Omicron variants tested, including XBB.1.5, EG.5.1, and BA.2.86, contrasting the diminished potency of parental antibodies delivered as monotherapies or as a cocktail. Furthermore, prophylactic delivery of CoV2-biRN5 significantly reduced the viral load within the lungs of K18-hACE2 mice following challenge with SARS-CoV-2 XBB.1.5. In conclusion, our NTD-RBD bsAbs offer promising potential for the design of resilient, next-generation antibody therapeutics against SARS-CoV-2 VOCs.

Transcription of HIV-1 at sites of intact latent provirus integration.

HIV-1 anti-retroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 10010,000X less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir thereby influencing cytopathic effects and proviral immune evasion.

Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Subvariant Neutralization Following a Primary Vaccine Series of NVX-CoV2373 and BNT162b2 Monovalent Booster Vaccine.

We evaluated the immunologic response to a novel vaccine regimen that included 2 doses of NVX-CoV2373 (Novavax) followed by 1 dose of BNT162b2 (Pfizer-BioNTech) monovalent booster vaccine. A durable neutralizing antibody response to Omicron BA.4/BA.5 and BA.1 variants was observed at month 6 after the booster, while immune escape was noted for the XBB.1.5 variant.

SARS-CoV-2 spike glycosylation affects function and neutralization sensitivity.

The glycosylation of viral envelope proteins can play important roles in virus biology and immune evasion. The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) includes 22 N-linked glycosylation sequons and 17 O-linked glycosites. We investigated the effect of individual glycosylation sites on SARS-CoV-2 S function in pseudotyped virus infection assays and on sensitivity to monoclonal and polyclonal neutralizing antibodies. In most cases, the removal of individual glycosylation sites decreased the infectiousness of the pseudotyped virus. For glycosylation mutants in the N-terminal domain and the receptor-binding domain (RBD), reduction in pseudotype infectivity was predicted by a commensurate reduction in the level of virion-incorporated S protein and reduced S trafficking to the cell surface. Notably, the presence of a glycan at position N343 within the RBD had diverse effects on neutralization by RBD-specific monoclonal antibodies cloned from convalescent individuals. The N343 glycan reduced the overall sensitivity to polyclonal antibodies in plasma from COVID-19 convalescent individuals, suggesting a role for SARS-CoV-2 S glycosylation in immune evasion. However, vaccination of convalescent individuals produced neutralizing activity that was resilient to the inhibitory effect of the N343 glycan.IMPORTANCEThe attachment of glycans to the spike proteins of viruses during their synthesis and movement through the secretory pathway can affect their properties. This study shows that the glycans attached to the severe acute respiratory syndrome coronavirus-2 spike protein enable its movement to the cell surface and incorporation into virus particles. Certain glycans, including one that is attached to asparagine 343 in the receptor-binding domain of the spike protein, can also affect virus neutralization by antibodies. This glycan can increase or decrease sensitivity to individual antibodies, likely through direct effects on antibody epitopes and modulation of spike conformation. However, the overall effect of the glycan in the context of the polyclonal mixture of antibodies in convalescent serum is to reduce neutralization sensitivity. Overall, this study highlights the complex effects of glycosylation on spike protein function and immune evasion.

Role of affinity in plasma cell development in the germinal center light zone.

Protective immune responses to many pathogens depend on the development of high-affinity antibody-producing plasma cells (PC) in germinal centers (GCs). Transgenic models suggest that there is a stringent affinity-based barrier to PC development. Whether a similar high-affinity barrier regulates PC development under physiologic circumstances and the nature of the PC fate decision has not been defined precisely. Here, we use a fate-mapping approach to examine the relationship between GC B cells selected to undergo additional rounds of affinity maturation, GC pre-PC, and PC. The data show that initial PC selection overlaps with GC B cell selection, but that the PC compartment accumulates a less diverse and higher affinity collection of antibodies over time. Thus, whereas the GC continues to diversify over time, affinity-based pre-PC selection sieves the GC to enable the accumulation of a more restricted group of high-affinity antibody-secreting PC.