Plasma neutralization properties of the SARS-CoV-2 Omicron variant

BACKGROUNDThe Omicron SARS-CoV-2 variant has spread internationally and is responsible for rapidly increasing case numbers. The emergence of divergent variants in the context of a heterogeneous and evolving neutralizing antibody response in host populations might compromise protection afforded by vaccines or prior infection. METHODSWe measured neutralizing antibody titers in 169 longitudinally collected plasma samples using pseudotypes bearing the Wuhan-hu-1 or the Omicron variant or a laboratory-designed neutralization-resistant SARS-CoV-2 spike (PMS20). Plasmas were obtained from convalescents who did or did not subsequently receive an mRNA vaccine, or naive individuals who received 3-doses of mRNA or 1-dose Ad26 vaccines. Samples were collected approximately 1, 5-6 and 12 months after initial vaccination or infection. RESULTSLike PMS20, the Omicron spike protein was substantially resistant to neutralization compared to Wuhan-hu-1. In convalescent plasma the median deficit in neutralizing activity against PMS20 or Omicron was 30- to 60-fold. Plasmas from recipients of 2 mRNA vaccine doses were 30- to 180- fold less potent against PMS20 and Omicron than Wuhan-hu-1. Notably, previously infected or two-mRNA dose vaccinated individuals who received additional mRNA vaccine dose(s) had 38 to 154-fold and 35 to 214-fold increases in neutralizing activity against Omicron and PMS20 respectively. CONCLUSIONSOmicron exhibits similar distribution of sequence changes and neutralization resistance as does a laboratory-designed neutralization-resistant spike protein, suggesting natural evolutionary pressure to evade the human antibody response. Currently available mRNA vaccine boosters, that may promote antibody affinity maturation, significantly ameliorate SARS-CoV-2 neutralizing antibody titers.

Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies

Antibodies elicited in response to infection undergo somatic mutation in germinal centers that can result in higher affinity for the cognate antigen. To determine the effects of somatic mutation on the properties of SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibodies, we analyzed six independent antibody lineages. As well as increased neutralization potency, antibody evolution changed pathways for acquisition of resistance and, in some cases, restricted the range of neutralization escape options. For some antibodies, maturation apparently imposed a requirement for multiple spike mutations to enable escape. For certain antibody lineages, maturation enabled neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.

Multimeric nanobodies from camelid engineered mice and llamas potently neutralize SARS-CoV-2 variants

Since the start of the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 2 million deaths worldwide. Multiple vaccines have been deployed to date, but the continual evolution of the viral receptor-binding domain (RBD) has recently challenged their efficacy. In particular, SARS-CoV-2 variants originating in the U.K. (B.1.1.7), South Africa (B.1.351) and New York (B.1.526) have reduced neutralization activity from convalescent sera and compromised the efficacy of antibody cocktails that received emergency use authorization. Whereas vaccines can be updated periodically to account for emerging variants, complementary strategies are urgently needed to avert viral escape. One potential alternative is the use of camelid VHHs (also known as nanobodies), which due to their small size can recognize protein crevices that are inaccessible to conventional antibodies. Here, we isolate anti-RBD nanobodies from llamas and "nanomice" we engineered to produce VHHs cloned from alpacas, dromedaries and camels. Through binding assays and cryo-electron microscopy, we identified two sets of highly neutralizing nanobodies. The first group expresses VHHs that circumvent RBD antigenic drift by recognizing a region outside the ACE2-binding site that is conserved in coronaviruses but is not typically targeted by monoclonal antibodies. The second group is almost exclusively focused to the RBD-ACE2 interface and fails to neutralize pseudoviruses carrying the E484K or N501Y substitutions. Notably however, they do neutralize the RBD variants when expressed as homotrimers, rivaling the most potent antibodies produced to date against SARS-CoV-2. These findings demonstrate that multivalent nanobodies overcome SARS-CoV-2 variant mutations through two separate mechanisms: enhanced avidity for the ACE2 binding domain, and recognition of conserved epitopes largely inaccessible to human antibodies. Therefore, while new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.

Increased Potency and Breadth of SARS-CoV-2 Neutralizing Antibodies After a Third mRNA Vaccine Dose

The omicron variant of SARS-CoV-2 infected very large numbers of SARS-CoV-2 vaccinated and convalescent individuals1-3. The penetrance of this variant in the antigen experienced human population can be explained in part by the relatively low levels of plasma neutralizing activity against Omicron in people who were infected or vaccinated with the original Wuhan-Hu-1 strain4-7. The 3rd mRNA vaccine dose produces an initial increase in circulating anti-Omicron neutralizing antibodies, but titers remain 10-20-fold lower than against Wuhan-Hu-1 and are, in many cases, insufficient to prevent infection7. Despite the reduced protection from infection, individuals that received 3 doses of an mRNA vaccine were highly protected from the more serious consequences of infection8. Here we examine the memory B cell repertoire in a longitudinal cohort of individuals receiving 3 mRNA vaccine doses9,10. We find that the 3rd dose is accompanied by an increase in, and evolution of, anti-receptor binding domain specific memory B cells. The increase is due to expansion of memory B cell clones that were present after the 2nd vaccine dose as well as the emergence of new clones. The antibodies encoded by these cells showed significantly increased potency and breadth when compared to antibodies obtained after the 2nd vaccine dose. Notably, the increase in potency was especially evident among newly developing clones of memory cells that differed from the persisting clones in targeting more conserved regions of the RBD. Overall, more than 50% of the analyzed neutralizing antibodies in the memory compartment obtained from individuals receiving a 3rd mRNA vaccine dose neutralized Omicron. Thus, individuals receiving 3 doses of an mRNA vaccine encoding Wuhan-Hu-1, have a diverse memory B cell repertoire that can respond rapidly and produce antibodies capable of clearing even diversified variants such as Omicron. These data help explain why a 3rd dose of an mRNA vaccine that was not specifically designed to protect against variants is effective against variant-induced serious disease.

Viral replication in human macrophages enhances an inflammatory cascade and interferon driven chronic COVID-19 in humanized mice.

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA, and sustained interferon (IFN) response all of which are recapitulated and required for pathology in the SARS-CoV-2 infected MISTRG6-hACE2 humanized mouse model of COVID-19 with a human immune system1-20. Blocking either viral replication with Remdesivir21-23 or the downstream IFN stimulated cascade with anti-IFNAR2 in vivo in the chronic stages of disease attenuated the overactive immune-inflammatory response, especially inflammatory macrophages. Here, we show SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release IL-1 and IL-18 and undergo pyroptosis thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and its accompanying inflammatory response is necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Remarkably, this same blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 by production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals

During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.