SARS-CoV-2 Delta derivatives impact on neutralization of Covishield recipient sera

The emergence of SARS-CoV-2 Delta variant and its derivatives has created grave public health problem worldwide. The high transmissibility associated with this variant has led to daily increase in the number of SARS-CoV-2 infections. Delta variant has slowly dominated the other variants of concern. Subsequently, Delta has further mutated to Delta AY.1 to Delta AY.126. Of these, Delta AY.1 has been reported from several countries including India and considered to be highly infectious and probable escape mutant. Considering the possible immune escape, we had already evaluated the efficacy of the BBV152 against Delta and Delta AY.1 variants. Here, we have evaluated the neutralizing potential of sera of COVID-19 naive vaccinees (CNV) immunized with two doses of vaccine, COVID-19 recovered cases immunized with two doses of vaccine (CRV) and breakthrough infections (BTI) post immunization with two doses of vaccine against Delta, Delta AY.1 and B.1.617.3 using 50% plaque reduction neutralization test (PRNT50). Our study observed low NAb titer in CNV group against all the variants compared to CRV and BTI groups. Delta variant has shown highest reduction of 27.3-fold in NAb titer among CNV group compared to other groups and variants. Anti-S1-RBD IgG immune response among all the groups was also substantiated with NAb response. Compromised neutralization was observed against Delta and Delta AY.1 compared B.1 in all three groups. However, it provided protection against severity of the disease and fatality.

Analysis of the Physicochemical Properties, Replication and Pathophysiology of a Massively Glycosylated Hepatitis B Virus HBsAg Escape Mutant

Mutations in HBsAg, the surface antigen of the hepatitis B virus (HBV), might affect the serum HBV DNA level of HBV-infected patients, since the reverse transcriptase (RT) domain of HBV polymerase overlaps with the HBsAg-coding region. We previously identified a diagnostic escape mutant (W3S) HBV that produces massively glycosylated HBsAg. In this study, we constructed an HBV-producing vector that expresses W3S HBs (pHB-W3S) along with a wild-type HBV-producing plasmid (pHB-WT) in order to analyze the physicochemical properties, replication, and antiviral drug response of the mutant. Transfection of either pHB-WT or W3S into HepG2 cells yielded similar CsCl density profiles and eAg expression, as did transfection of a glycosylation defective mutant, pHB-W3S (N146G), in which a glycosylation site at the 146aa asparagine (N) site of HBs was mutated to glycine (G). Virion secretion, however, seemed to be severely impaired in cases of pHB-W3S and pHB-W3S (N146G), compared with pHB-WT, as determined by qPCR and Southern blot analysis. Furthermore, inhibition of glycosylation using tunicamycinTM on wild-type HBV production also reduced the virion secretion. These results suggested that the HBV core and Dane particle could be formed either by massively glycosylated or glycosylation-defective HBsAg, but reduced and/or almost completely blocked the virion secretion efficiency, indicating that balanced glycosylation of HBsAg is required for efficient release of HBV, and mutations inducing an imbalanced glycosylation of HBs would cause the virion to become stuck in the cells, which might be associated with various pathogeneses due to HBV infection.

Potent and protective IGHV3-53/3-66 public antibodies and their shared escape mutant on the spike of SARS-CoV-2

Neutralizing antibodies (nAbs) to SARS-CoV-2 hold powerful potentials for clinical interventions against COVID-19 disease. However, their common genetic and biologic features remain elusive. Here we interrogate a total of 165 antibodies from eight COVID-19 patients, and find that potent nAbs from different patients have disproportionally high representation of IGHV3-53/3-66 usage, and therefore termed as public antibodies. Crystal structural comparison of these antibodies reveals they share similar angle of approach to RBD, overlap in buried surface and binding residues on RBD, and have substantial spatial clash with receptor angiotensin-converting enzyme-2 (ACE2) in binding to RBD. Site-directed mutagenesis confirms these common binding features although some minor differences are found. One representative antibody, P5A-3C8, demonstrates extraordinarily protective efficacy in a golden Syrian hamster model against SARS-CoV-2 infection. However, virus escape analysis identifies a single natural mutation in RBD, namely K417N found in B.1.351 variant from South Africa, abolished the neutralizing activity of these public antibodies. The discovery of public antibodies and shared escape mutation highlight the intricate relationship between antibody response and SARS-CoV-2, and provide critical reference for the development of antibody and vaccine strategies to overcome the antigenic variation of SARS-CoV-2.

Targeted vaccination and the speed of SARS-CoV-2 adaptation

The limited supply of vaccines against SARS-CoV-2 raises the question of targeted vaccination. Older and more sensitive hosts should be vaccinated first to minimize the disease burden. But what are the evolutionary consequences of targeted vaccination? We clarify the consequences of different vaccination strategies through the analysis of the speed of viral adaptation measured as the rate of change of the frequency of vaccine-escape mutations. We show that a vaccine-escape mutant is expected to spread faster if vaccination targets individuals which are likely to be involved in a higher number of contacts. We also discuss the pros and cons of dose-sparing strategies. Because delaying the second dose increases the proportion of the population vaccinated with a single dose, this strategy can both speed-up the spread of the vaccine-escape mutant and reduce the cumulated number of deaths. Hence, slowing down viral adaptation may not always be the optimal vaccination strategy. We contend that a careful assessment of the consequences of alternative vaccination strategies on both (i) the speed of adaptation and (ii) the mortality is required to determine which individuals should be vaccinated first.

Cross-reactive TCR with alloreactivity for immunodominant HIV-1 epitope Gag TL9 with enhanced control of viral infection

Although both HLA B*81:01 and HLA B*42:01 are members of the B7 supertype and can present many of the same HIV-1 epitopes, the identification of a dual-reactive T cell phenotype was unexpected, since structural data suggested that TL9 peptide binds to each allele in a distinct conformation. How the dual-reactive TCR recognizes these radically distinct p-MHC surfaces is revealed by our structural study, that the introduction of TCR T18A induces a molecular switch of the TL9 peptide in B4201 to approach its conformation in B8101. Most importantly, unique docking of CDR3β towards MHC but not peptide ligand strengthens the peptide tolerance of T18A, extends the ability of TCR to adapt mutations. Moreover, the high affinity of dual active TCR for WT and escape mutant TL9 highlights the functional advantage of the alloreactive phenotype.

The `greed ́ and `nationalism ́ in vaccination against Covid-19 lead to the Tragedy of the Commons, Nash equilibrium and Giffen's goods, giving SARS-CoV-2 more opportunities to escape mutants.

It is possible to follow an ideal vaccination global strategy, that will reduce to the minimum the number of infected and dead by COVID-19 as well as the likelihood of an escape mutant. This ideal vaccination strategy needs of a high degree of international cooperation among different countries. International initiatives as COVAX, advocated by the WHO, try to achieve such goal. However, in the present scenario with limited number of vaccine doses, Game Theory model also predicts that they are becoming goods subject to the Tragedy of the Commons. Countries with the capability to control vaccine dose distribution will desert from the international agreements, vaccinating first to their own nationals. If such behaviour is known by other countries (as is happening now) more countries will be tempted to desert as well. Eventually it will end in a Nash equilibrium, which stops the ideal vaccination strategy. Furthermore, in poorer countries, with great difficulties to get access to vaccine doses, these will be transformed into Giffen goods. Vaccination data available to date, show that we are in a scenario where vaccine doses have transformed into goods subject to the Tragedy of the Commons, countries desert from cooperation reaching a Nash equilibrium and vaccine doses are close to become a Giffen good for the poorer countries. This dynamic will not only rise the number of infected and dead by COVID-19, but also the likelihood of an escape mutant (in the SARS-CoV-2 populations) will be increased. If, eventually, this turns out to be the new scenario new vaccines could be developed for those new escape mutant strains, going back to a very similar setting as the starting one. Most likely, the world will reach a Pareto optimality (equilibrium between desertion and cooperation) due the pressure of those who seek high levels of cooperation to attain a global vaccination strategy.

Novel epitopes of human monoclonal antibodies targeting the influenza virus N1 neuraminidase

Influenza virus neuraminidase (NA) targeting antibodies are an independent correlate of protection against infection. Antibodies against the NA act by blocking enzymatic activity, preventing virus release and transmission. As we advance the development of improved influenza virus vaccines that incorporate standard amounts of NA antigen, it is important to identify the antigenic targets of human monoclonal antibodies (mAbs). Additionally, it is important to understand how escape from mAbs changes viral fitness. Here, we describe escape mutants generated by serial passage of A/Netherlands/602/2009 (H1N1) in the presence of human anti-N1 mAbs. We observed escape mutations on the N1 protein around the enzymatic site (S364N, N369T and R430Q) and also detected escape mutations located on the sides and bottom of the NA (N88D, N270D and Q313K/R). We found that a majority of escape mutant viruses had increased fitness in vitro but not in vivo. This work increases our understanding of how human antibody responses target the N1 protein.

Mutations in the Hemagglutinin Stalk Domain Do Not Permit Escape from a Protective, Stalk-Based Vaccine-Induced Immune Response in the Mouse Model

ABSTRACT Current seasonal influenza virus vaccines target regions of the hemagglutinin (HA) head domain that undergo constant antigenic change, forcing the painstaking annual reformulation of vaccines. The development of broadly protective or universal influenza virus vaccines that induce cross-reactive, protective immune responses could circumvent the need to reformulate current seasonal vaccines. Many of these vaccine candidates target the HA stalk domain, which displays epitopes conserved within and across influenza virus subtypes, including those with pandemic potential. While HA head-mediated antigenic drift is well understood, the potential for antigenic drift in the stalk domain is understudied. Using a panel of HA stalk-specific monoclonal antibodies (MAbs), we applied selection pressure to the stalk domain of A/Netherlands/602/2009 (pdmH1N1) to determine fitness and phenotypes of escape mutant viruses (EMVs). We found that HA stalk MAbs with lower cross-reactivity caused single HA stalk escape mutations, whereas MAbs with broader cross-reactivity forced multiple mutations in the HA. Each escape mutant virus greatly decreased mAb neutralizing activity, but escape mutations did not always ablate MAb binding or Fc-Fc receptor-based effector functions. Escape mutant viruses were not attenuated in vitro but showed attenuation in an in vivo mouse model. Importantly, mice vaccinated with a chimeric HA universal vaccine candidate were protected from lethal challenge with EMVs despite these challenge viruses containing escape mutations in the stalk domain. Our study indicates that while the HA stalk domain can mutate under strong MAb selection pressure, mutant viruses may have attenuated phenotypes and do not evade a polyclonal, stalk-based vaccine-induced response. IMPORTANCE Broadly protective or universal influenza virus vaccines target viral epitopes that appear to be conserved. However, it is unclear whether the virus will be able to escape once immunological pressure is applied to these epitopes through vaccination of large proportions of the population. Studies that investigate the fitness and antigenic characteristics of viruses that escape immunological pressure on these conserved epitopes are therefore urgently needed.

Monoclonal antibodies targeting the influenza virus N6 neuraminidase

Influenza A viruses are a diverse species that include 16 hemagglutinin (HA) subtypes and 9 neuraminidase (NA) subtypes. While the antigenicity of many HA subtypes is reasonably well studied, less is known about NA antigenicity, especially when it comes to non-human subtypes that only circulate in animal reservoirs. The N6 NA subtypes are mostly found in viruses infecting birds. However, they have also been identified in viruses that infect mammals, such as swine and seals. More recently, highly pathogenic H5N6 subtype viruses have caused rare infections and mortality in humans. Here, we generated murine mAbs to the N6 NA, characterized their breadth and antiviral properties in vitro and in vivo and mapped their epitopes by generating escape mutant viruses. We found that the antibodies had broad reactivity across the American and Eurasian N6 lineages, but relatively little binding and inhibition of the H5N6 NA. Several of the antibodies exhibited strong NA inhibition activity and some also showed activity in the antibody dependent cellular cytotoxicity reporter assay and neutralization assay. In addition, we generated escape mutant viruses for six monoclonal antibodies and found mutations on the lateral ridge of the NA. Lastly, we observed variable protection in H4N6 and H5N6 mouse challenge models when the antibodies were given prophylactically.ImportanceThe N6 NA has recently gained prominence due to the emergence of highly pathogenic H5N6 viruses. Currently, there is limited characterization of the antigenicity of avian N6 neuraminidase. Our data is an important first step towards a better understanding of the N6 NA antigenicity.