B cell genomics behind cross-neutralization of SARS-CoV-2 variants and SARS-CoV.

Monoclonal antibodies (mAbs) are a focus in vaccine and therapeutic design to counteract severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. Here, we combined B cell sorting with single-cell VDJ and RNA sequencing (RNA-seq) and mAb structures to characterize B cell responses against SARS-CoV-2. We show that the SARS-CoV-2-specific B cell repertoire consists of transcriptionally distinct B cell populations with cells producing potently neutralizing antibodies (nAbs) localized in two clusters that resemble memory and activated B cells. Cryo-electron microscopy structures of selected nAbs from these two clusters complexed with SARS-CoV-2 spike trimers show recognition of various receptor-binding domain (RBD) epitopes. One of these mAbs, BG10-19, locks the spike trimer in a closed conformation to potently neutralize SARS-CoV-2, the recently arising mutants B.1.1.7 and B.1.351, and SARS-CoV and cross-reacts with heterologous RBDs. Together, our results characterize transcriptional differences among SARS-CoV-2-specific B cells and uncover cross-neutralizing Ab targets that will inform immunogen and therapeutic design against coronaviruses.

Recurrent Potent Human Neutralizing Antibodies to Zika Virus in Brazil and Mexico.

Antibodies to Zika virus (ZIKV) can be protective. To examine the antibody response in individuals who develop high titers of anti-ZIKV antibodies, we screened cohorts in Brazil and Mexico for ZIKV envelope domain III (ZEDIII) binding and neutralization. We find that serologic reactivity to dengue 1 virus (DENV1) EDIII before ZIKV exposure is associated with increased ZIKV neutralizing titers after exposure. Antibody cloning shows that donors with high ZIKV neutralizing antibody titers have expanded clones of memory B cells that express the same immunoglobulin VH3-23/VK1-5 genes. These recurring antibodies cross-react with DENV1, but not other flaviviruses, neutralize both DENV1 and ZIKV, and protect mice against ZIKV challenge. Structural analyses reveal the mechanism of recognition of the ZEDIII lateral ridge by VH3-23/VK1-5 antibodies. Serologic testing shows that antibodies to this region correlate with serum neutralizing activity to ZIKV. Thus, high neutralizing responses to ZIKV are associated with pre-existing reactivity to DENV1 in humans.

Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes.

Increased immune evasion by SARS-CoV-2 variants of concern highlights the need for new therapeutic neutralizing antibodies. Immunization with nanoparticles co-displaying spike receptor-binding domains (RBDs) from eight sarbecoviruses (mosaic-8 RBD-nanoparticles) efficiently elicits cross-reactive polyclonal antibodies against conserved sarbecovirus RBD epitopes. Here, we identified monoclonal antibodies (mAbs) capable of cross-reactive binding and neutralization of animal sarbecoviruses and SARS-CoV-2 variants by screening single mouse B cells secreting IgGs that bind two or more sarbecovirus RBDs. Single-particle cryo-EM structures of antibody-spike complexes, including a Fab-Omicron complex, mapped neutralizing mAbs to conserved class 1/4 RBD epitopes. Structural analyses revealed neutralization mechanisms, potentials for intra-spike trimer cross-linking by IgGs, and induced changes in trimer upon Fab binding. In addition, we identified a mAb-resembling Bebtelovimab, an EUA-approved human class 3 anti-RBD mAb. These results support using mosaic RBD-nanoparticle vaccination to generate and identify therapeutic pan-sarbecovirus and pan-variant mAbs.

Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques.

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires.

Structural basis for Zika envelope domain III recognition by a germline version of a recurrent neutralizing antibody.

Recent epidemics demonstrate the global threat of Zika virus (ZIKV), a flavivirus transmitted by mosquitoes. Although infection is usually asymptomatic or mild, newborns of infected mothers can display severe symptoms, including neurodevelopmental abnormalities and microcephaly. Given the large-scale spread, symptom severity, and lack of treatment or prophylaxis, a safe and effective ZIKV vaccine is urgently needed. However, vaccine design is complicated by concern that elicited antibodies (Abs) may cross-react with other flaviviruses that share a similar envelope protein, such as dengue virus, West Nile virus, and yellow fever virus. This cross-reactivity may worsen symptoms of a subsequent infection through Ab-dependent enhancement. To better understand the neutralizing Ab response and risk of Ab-dependent enhancement, further information on germline Ab binding to ZIKV and the maturation process that gives rise to potently neutralizing Abs is needed. Here we use binding and structural studies to compare mature and inferred-germline Ab binding to envelope protein domain III of ZIKV and other flaviviruses. We show that affinity maturation of the light-chain variable domain is important for strong binding of the recurrent VH3-23/VK1-5 neutralizing Abs to ZIKV envelope protein domain III, and identify interacting residues that contribute to weak, cross-reactive binding to West Nile virus. These findings provide insight into the affinity maturation process and potential cross-reactivity of VH3-23/VK1-5 neutralizing Abs, informing precautions for protein-based vaccines designed to elicit germline versions of neutralizing Abs.

A combination of two human monoclonal antibodies limits fetal damage by Zika virus in macaques.

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement (ADE) in vitro and extend their half-lives. Here we report on prophylactic coadministration of the Fc-modified antibodies to pregnant rhesus macaques challenged three times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission, protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV.

Designed oligomers of cyanovirin-N show enhanced HIV neutralization.

Cyanovirin-N (CV-N) is a small, cyanobacterial lectin that neutralizes many enveloped viruses, including human immunodeficiency virus type I (HIV-1). This antiviral activity is attributed to two homologous carbohydrate binding sites that specifically bind high mannose glycosylation present on envelope glycoproteins such as HIV-1 gp120. We created obligate CV-N oligomers to determine whether increasing the number of binding sites has an effect on viral neutralization. A tandem repeat of two CV-N molecules (CVN(2)) increased HIV-1 neutralization activity by up to 18-fold compared to wild-type CV-N. In addition, the CVN(2) variants showed extensive cross-clade reactivity and were often more potent than broadly neutralizing anti-HIV antibodies. The improvement in activity and broad cross-strain HIV neutralization exhibited by these molecules holds promise for the future therapeutic utility of these and other engineered CV-N variants.

Designed mosaic nanoparticles enhance cross-reactive immune responses in mice.

1Using computational methods, we designed 60-mer nanoparticles displaying SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs) by (i) creating RBD sequences with 6 mutations in the SARS-COV-2 WA1 RBD that were predicted to retain proper folding and abrogate antibody responses to variable epitopes (mosaic-2COMs; mosaic-5COM), and (ii) selecting 7 natural sarbecovirus RBDs (mosaic-7COM). These antigens were compared with mosaic-8b, which elicits cross-reactive antibodies and protects from sarbecovirus challenges in animals. Immunizations in naïve and COVID-19 pre-vaccinated mice revealed that mosaic-7COM elicited higher binding and neutralization titers than mosaic-8b and related antigens. Deep mutational scanning showed that mosaic-7COM targeted conserved RBD epitopes. Mosaic-2COMs and mosaic-5COM elicited higher titers than homotypic SARS-CoV-2 Beta RBD-nanoparticles and increased potencies against some SARS-CoV-2 variants than mosaic-7COM. However, mosaic-7COM elicited more potent responses against zoonotic sarbecoviruses and highly mutated Omicrons. These results support using mosaic-7COM to protect against highly mutated SARS-CoV-2 variants and zoonotic sarbecoviruses with spillover potential.

Proactive vaccination using multiviral Quartet Nanocages to elicit broad anti-coronavirus responses.

Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle's branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 'Kraken' RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection.

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.

Mosaic sarbecovirus nanoparticles elicit cross-reactive responses in pre-vaccinated animals.

Immunization with mosaic-8b [60-mer nanoparticles presenting 8 SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs)] elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate-mapping in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19 vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.

Cross-sectional survey in Central African Republic finds mortality 4-times higher than UN statistics: how can we not know the Central African Republic is in such an acute humanitarian crisis?

BACKGROUND: CAR is one of the poorest countries in the world. While UN statistics suggest that there is no health emergency in the country, two recently published mortality surveys contradict this. Moreover, recent accusations of massive scale human rights abuses by mercenaries suggested the need for a nationwide mortality survey. METHODS: Two stage cluster surveys were conducted in two different strata: one in the roughly half of the country within the Government's control, and one in the areas mostly outside of the Government's control. We randomly selected 40 clusters of 10 households in each stratum. The survey included questions on vital events with open-ended questions about health and household challenges at the beginning and end of each interview. RESULTS: 70 of 80 selected clusters were successfully visited. We interviewed 699 households, containing 5070 people. 11 households (1.6%) refused to be interviewed and approximately 18.3% of households were absent at the time of visitation, mainly in the safer Government controlled areas. Interviewed households had a birth rate of 42.6 /1000 / year (95%CI 35.4-59.7) and a crude mortality rate (CMR) of 1.57 /10,000/day (95%CI: 1.36-1.78). The birth rate was lower and the death rate markedly higher in the strata outside of Government control. Families described malaria or fever, and diarrhea as the primary reported causes of death with violence accounting for 6% of all deaths. CONCLUSIONS: CAR is experiencing a severe health emergency, with the highest measured nationwide mortality in the world to our knowledge. UN published death rate estimates appear to be less than one fourth of reality. There is a desperate need for food aid in the form of general distributions in CAR, along with the accompanying work programs, seed and tool distributions needed to restart local economies. This is of particular importance in rural areas outside of the Government control. While some humanitarian actors are doing their best to respond, the crisis level mortality rate suggests that the needs in CAR are being largely unmet.

A combination of two resistance mechanisms is critical for tick-borne encephalitis virus escape from a broadly neutralizing human antibody.

Tick-borne encephalitis virus (TBEV) is a flavivirus that causes human neuroinfections and represents a growing health problem. The human monoclonal antibody T025 targets envelope protein domain III (EDIII) of TBEV and related tick-borne flaviviruses, potently neutralizing TBEV in vitro and in preclinical models, representing a promising candidate for clinical development. We demonstrate that TBEV escape in the presence of T025 or T028 (another EDIII-targeting human monoclonal antibody) results in virus variants of reduced pathogenicity, characterized by distinct sets of amino acid changes in EDII and EDIII that are jointly needed to confer resistance. EDIII substitution K311N impairs formation of a salt bridge critical for T025-epitope interaction. EDII substitution E230K is not on the T025 epitope but likely induces quaternary rearrangements of the virus surface because of repulsion of positively charged residues on the adjacent EDI. A combination of T025 and T028 prevents virus escape and improves neutralization.

Multiviral Quartet Nanocages Elicit Broad Anti-Coronavirus Responses for Proactive Vaccinology.

Defending against future pandemics may require vaccine platforms that protect across a range of related pathogens. The presentation of multiple receptor-binding domains (RBDs) from evolutionarily-related viruses on a nanoparticle scaffold elicits a strong antibody response to conserved regions. Here we produce quartets of tandemly-linked RBDs from SARS-like betacoronaviruses coupled to the mi3 nanocage through a SpyTag/SpyCatcher spontaneous reaction. These Quartet Nanocages induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented on the vaccine. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increased the strength and breadth of an otherwise narrow immune response. Quartet Nanocages are a strategy with potential to confer heterotypic protection against emergent zoonotic coronavirus pathogens and facilitate proactive pandemic protection.

Structural basis for a conserved neutralization epitope on the receptor-binding domain of SARS-CoV-2.

Antibody-mediated immunity plays a crucial role in protection against SARS-CoV-2 infection. We isolated a panel of neutralizing anti-receptor-binding domain (RBD) antibodies elicited upon natural infection and vaccination and showed that they recognize an immunogenic patch on the internal surface of the core RBD, which faces inwards and is hidden in the "down" state. These antibodies broadly neutralize wild type (Wuhan-Hu-1) SARS-CoV-2, Beta and Delta variants and some are effective against other sarbecoviruses. We observed a continuum of partially overlapping antibody epitopes from lower to upper part of the inner face of the RBD and some antibodies extend towards the receptor-binding motif. The majority of antibodies are substantially compromised by three mutational hotspots (S371L/F, S373P and S375F) in the lower part of the Omicron BA.1, BA.2 and BA.4/5 RBD. By contrast, antibody IY-2A induces a partial unfolding of this variable region and interacts with a conserved conformational epitope to tolerate all antigenic variations and neutralize diverse sarbecoviruses as well. This finding establishes that antibody recognition is not limited to the normal surface structures on the RBD. In conclusion, the delineation of functionally and structurally conserved RBD epitopes highlights potential vaccine and therapeutic candidates for COVID-19.

CD4 binding site immunogens elicit heterologous anti-HIV-1 neutralizing antibodies in transgenic and wild-type animals.

Passive transfer of broadly neutralizing anti-HIV-1 antibodies (bNAbs) protects against infection, and therefore, eliciting bNAbs by vaccination is a major goal of HIV-1 vaccine efforts. bNAbs that target the CD4 binding site (CD4bs) on HIV-1 Env are among the most broadly active, but to date, responses elicited against this epitope in vaccinated animals have lacked potency and breadth. We hypothesized that CD4bs bNAbs resembling the antibody IOMA might be easier to elicit than other CD4bs antibodies that exhibit higher somatic mutation rates, a difficult-to-achieve mechanism to accommodate Env's N276gp120 N-glycan, and rare five-residue light chain complementarity-determining region 3. As an initial test of this idea, we developed IOMA germline-targeting Env immunogens and evaluated a sequential immunization regimen in transgenic mice expressing germline-reverted IOMA. These mice developed CD4bs epitope-specific responses with heterologous neutralization, and cloned antibodies overcame neutralization roadblocks, including accommodating the N276gp120 glycan, with some neutralizing selected HIV-1 strains more potently than IOMA. The immunization regimen also elicited CD4bs-specific responses in mice containing polyclonal antibody repertoires as well as rabbits and rhesus macaques. Thus, germline targeting of IOMA-class antibody precursors represents a potential vaccine strategy to induce CD4bs bNAbs.

Mosaic RBD nanoparticles protect against multiple sarbecovirus challenges in animal models.

To combat future SARS-CoV-2 variants and spillovers of SARS-like betacoronaviruses (sarbecoviruses) threatening global health, we designed mosaic nanoparticles presenting randomly-arranged sarbecovirus spike receptor-binding domains (RBDs) to elicit antibodies against conserved/relatively-occluded, rather than variable/immunodominant/exposed, epitopes. We compared immune responses elicited by mosaic-8 (SARS-CoV-2 and seven animal sarbecoviruses) and homotypic (only SARS-CoV-2) RBD-nanoparticles in mice and macaques, observing stronger responses elicited by mosaic-8 to mismatched (not on nanoparticles) strains including SARS-CoV and animal sarbecoviruses. Mosaic-8 immunization showed equivalent neutralization of SARS-CoV-2 variants including Omicron and protected from SARS-CoV-2 and SARS-CoV challenges, whereas homotypic SARS-CoV-2 immunization protected only from SARS-CoV-2 challenge. Epitope mapping demonstrated increased targeting of conserved epitopes after mosaic-8 immunization. Together, these results suggest mosaic-8 RBD-nanoparticles could protect against SARS-CoV-2 variants and future sarbecovirus spillovers.

Mosaic RBD nanoparticles protect against challenge by diverse sarbecoviruses in animal models.

To combat future severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and spillovers of SARS-like betacoronaviruses (sarbecoviruses) threatening global health, we designed mosaic nanoparticles that present randomly arranged sarbecovirus spike receptor-binding domains (RBDs) to elicit antibodies against epitopes that are conserved and relatively occluded rather than variable, immunodominant, and exposed. We compared immune responses elicited by mosaic-8 (SARS-CoV-2 and seven animal sarbecoviruses) and homotypic (only SARS-CoV-2) RBD nanoparticles in mice and macaques and observed stronger responses elicited by mosaic-8 to mismatched (not on nanoparticles) strains, including SARS-CoV and animal sarbecoviruses. Mosaic-8 immunization showed equivalent neutralization of SARS-CoV-2 variants, including Omicrons, and protected from SARS-CoV-2 and SARS-CoV challenges, whereas homotypic SARS-CoV-2 immunization protected only from SARS-CoV-2 challenge. Epitope mapping demonstrated increased targeting of conserved epitopes after mosaic-8 immunization. Together, these results suggest that mosaic-8 RBD nanoparticles could protect against SARS-CoV-2 variants and future sarbecovirus spillovers.

Use of and barriers to maternal health services in southeast Chad: results of a population-based survey 2019.

OBJECTIVES: Chad reports the second highest maternal mortality worldwide. We conducted a survey in Sila region in southeast Chad to estimate the use of maternal health services (MHS) and to identify barriers to access MHS. DESIGN: Retrospective cross-sectional, population-based survey using two-stage cluster sampling methodology. The survey consisted of two strata, Koukou Angarana and Goz Beida district in Sila region. We conducted systematic random sampling proportional to population size to select settlements in each strata in the first sampling stage; and in the second stage we selected households in the settlements using random walk procedure. We calculated survey-design-weighted proportions with 95% CIs. We performed univariate analysis and multivariable logistic regression to identify impact factors associated with the use of MHS. SETTING: We interviewed women in selected households in Sila region in 2019. PARTICIPANTS: Women at reproductive age, who have given birth in the previous 2 years and are living in Koukou Angarana and Goz Beida district. PRIMARY OUTCOMES: Use of and access barriers to MHS including antenatal care (ANC), delivery care in a health facility (DC), postnatal care (PNC) and contraceptive methods. RESULTS: In total, 624 women participated. Median age was 28 years, 95.4% were illiterate and 95.7% married. Use of ANC, DC and PNC was reported by 57.6% (95% CI: 49.3% to 65.5%), 22.5% (95% CI: 15.7% to 31.1%) and 32.9% (95% CI: 25.8% to 40.9%), respectively. Use of MHS was lower in rural compared with urban settings. Having attended ANC increased the odds of using DC by 4.3 (1.5-12.2) and using PNC by 6.4 (3.7-11.1). Factors related to transport and to culture and belief were the most frequently stated access barriers to MHS. CONCLUSION: In Sila region, use of MHS is low and does not meet WHO-defined standards regarding maternal health. Among all services, use of ANC was better than for other MHS. ANC usage is positively associated with the use of further life-saving MHS including DC and could be used as an entry point to the community. To increase use of MHS, interventions should include infrastructural improvements as well as community-based approaches to overcome access barriers related to culture and belief.

Antibody elicited by HIV-1 immunogen vaccination in macaques displaces Env fusion peptide and destroys a neutralizing epitope.

HIV-1 vaccine design aims to develop an immunogen that elicits broadly neutralizing antibodies against a desired epitope, while eliminating responses to off-target regions of HIV-1 Env. We report characterization of Ab1245, an off-target antibody against the Env gp120-gp41 interface, from V3-glycan patch immunogen-primed and boosted macaques. A 3.7 Å cryo-EM structure of an Ab1245-Env complex reveals one Ab1245 Fab binding asymmetrically to Env trimer at the gp120-gp41 interface using its long CDRH3 to mimic regions of gp41. The mimicry includes positioning of a CDRH3 methionine into the gp41 tryptophan clasp, resulting in displacement of the fusion peptide and fusion peptide-proximal region. Despite fusion peptide displacement, Ab1245 is non-neutralizing even at high concentrations, raising the possibility that only two fusion peptides per trimer are required for viral-host membrane fusion. These structural analyses facilitate immunogen design to prevent elicitation of Ab1245-like antibodies that block neutralizing antibodies against the fusion peptide.