Safety and immunogenicity of mRNA-based seasonal influenza vaccines formulated to include multiple A/H3N2 strains with or without the B/Yamagata strain in US adults aged 50-75 years: a phase 1/2, open-label, randomised trial.

Background: Inclusion of additional influenza A/H3N2 strains in seasonal influenza vaccines could expand coverage against multiple, antigenically distinct, cocirculating A/H3N2 clades and potentially replace the no longer circulating B/Yamagata strain. We aimed to evaluate the safety and immunogenicity of three next-generation seasonal influenza mRNA vaccines with different compositions that encode for haemagglutinins of multiple A/H3N2 strains, with or without the B/Yamagata strain, in adults.

Methods: This randomised, open-label, phase 1/2 trial enrolled healthy adults aged 50-75 years across 22 sites in the USA.

Enhancing antibody affinity through experimental sampling of non-deleterious CDR mutations predicted by machine learning.

The application of machine learning (ML) models to optimize antibody affinity to an antigen is gaining prominence. Unfortunately, the small and biased nature of the publicly available antibody-antigen interaction datasets makes it challenging to build an ML model that can accurately predict binding affinity changes due to mutations (ΔΔG). Recognizing these inherent limitations, we reformulated the problem to ask whether an ML model capable of classifying deleterious vs non-deleterious mutations can guide antibody affinity maturation in a practical setting.

Optimizing Antibody Affinity and Developability Using a Framework-CDR Shuffling Approach-Application to an Anti-SARS-CoV-2 Antibody.

The computational methods used for engineering antibodies for clinical development have undergone a transformation from three-dimensional structure-guided approaches to artificial-intelligence- and machine-learning-based approaches that leverage the large sequence data space of hundreds of millions of antibodies generated by next-generation sequencing (NGS) studies. Building on the wealth of available sequence data, we implemented a computational shuffling approach to antibody components, using the complementarity-determining region (CDR) and the framework region (FWR) to optimize an antibody for improved affinity and developability. This approach uses a set of rules to suitably combine the CDRs and FWRs derived from naturally occurring antibody sequences to engineer an antibody with high affinity and specificity.

Orthogonal immunoassays for IgG antibodies to SARS-CoV-2 antigens reveal that immune response lasts beyond 4 mo post illness onset.

Immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during the current pandemic remains a field of immense interest and active research worldwide. Although the severity of acute infection may depend on the intensity of innate and adaptive immunity, leading to higher morbidity and mortality, the longevity of IgG antibodies, including neutralizing activity to SARS-CoV-2, is viewed as a key correlate of immune protection. Amid reports and concern that there is a rapid decay of IgG antibody levels within 1 mo to 2 mo after acute infection, we set out to study the pattern and duration of IgG antibody response to various SARS-CoV-2 antigens in asymptomatic and symptomatic patients in a community setting.

The soft palate is an important site of adaptation for transmissible influenza viruses.

Influenza A viruses pose a major public health threat by causing seasonal epidemics and sporadic pandemics. Their epidemiological success relies on airborne transmission from person to person; however, the viral properties governing airborne transmission of influenza A viruses are complex. Influenza A virus infection is mediated via binding of the viral haemagglutinin (HA) to terminally attached α2,3 or α2,6 sialic acids on cell surface glycoproteins.

Glycan receptor specificity as a useful tool for characterization and surveillance of influenza A virus.

Influenza A viruses are rapidly evolving pathogens with the potential for novel strains to emerge and result in pandemic outbreaks in humans. Some avian-adapted subtypes have acquired the ability to bind to human glycan receptors and cause severe infections in humans but have yet to adapt to and transmit between humans. The emergence of new avian strains and their ability to infect humans has confounded their distinction from circulating human virus strains through linking receptor specificity to human adaptation.

Receptor specificity does not affect replication or virulence of the 2009 pandemic H1N1 influenza virus in mice and ferrets.

Human influenza viruses predominantly bind α2,6 linked sialic acid (SA) while avian viruses bind α2,3 SA-containing complex glycans. Virulence and tissue tropism of influenza viruses have been ascribed to this binding preference. We generated 2009 pandemic H1N1 (pH1N1) viruses with either predominant α2,3 or α2,6 SA binding and evaluated these viruses in mice and ferrets.

Glycan receptor binding of the influenza A virus H7N9 hemagglutinin.

The advent of H7N9 in early 2013 is of concern for a number of reasons, including its capability to infect humans, the lack of clarity in the etiology of infection, and because the human population does not have pre-existing immunity to the H7 subtype. Earlier sequence analyses of H7N9 hemagglutinin (HA) point to amino acid changes that predicted human receptor binding and impinge on the antigenic characteristics of the HA. Here, we report that the H7N9 HA shows limited binding to human receptors; however, should a single amino acid mutation occur, this would result in structural changes within the receptor binding site that allow for extensive binding to human receptors present in the upper respiratory tract.

Structural determinants for naturally evolving H5N1 hemagglutinin to switch its receptor specificity.

Of the factors governing human-to-human transmission of the highly pathogenic avian-adapted H5N1 virus, the most critical is the acquisition of mutations on the viral hemagglutinin (HA) to "quantitatively switch" its binding from avian to human glycan receptors. Here, we describe a structural framework that outlines a necessary set of H5 HA receptor-binding site (RBS) features required for the H5 HA to quantitatively switch its preference to human receptors. We show here that the same RBS HA mutations that lead to aerosol transmission of A/Vietnam/1203/04 and A/Indonesia/5/05 viruses, when introduced in currently circulating H5N1, do not lead to a quantitative switch in receptor preference.

N-linked glycosylation of the hemagglutinin protein influences virulence and antigenicity of the 1918 pandemic and seasonal H1N1 influenza A viruses.

The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic influenza virus; however, it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis, we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head.

Quantitative characterization of glycan-receptor binding of H9N2 influenza A virus hemagglutinin.

Avian influenza subtypes such as H5, H7 and H9 are yet to adapt to the human host so as to establish airborne transmission between humans. However, lab-generated reassorted viruses possessing hemagglutinin (HA) and neuraminidase (NA) genes from an avian H9 isolate and other genes from a human-adapted (H3 or H1) subtype acquired two amino acid changes in HA and a single amino acid change in NA that confer respiratory droplet transmission in ferrets. We previously demonstrated for human-adapted H1, H2 and H3 subtypes that quantitative binding affinity of their HA to α2→6 sialylated glycan receptors correlates with respiratory droplet transmissibility of the virus in ferrets.

Quantitative description of glycan-receptor binding of influenza A virus H7 hemagglutinin.

In the context of recently emerged novel influenza strains through reassortment, avian influenza subtypes such as H5N1, H7N7, H7N2, H7N3 and H9N2 pose a constant threat in terms of their adaptation to the human host. Among these subtypes, it was recently demonstrated that mutations in H5 and H9 hemagglutinin (HA) in the context of lab-generated reassorted viruses conferred aerosol transmissibility in ferrets (a property shared by human adapted viruses). We previously demonstrated that the quantitative binding affinity of HA to α2→6 sialylated glycans (human receptors) is one of the important factors governing human adaptation of HA.

Glycosylation at Asn91 of H1N1 haemagglutinin affects binding to glycan receptors.

The glycoprotein HA (haemagglutinin) on the surface of influenza A virus plays a central role in recognition and binding to specific host cell-surface glycan receptors and in fusion of viral membrane to the host nuclear membrane during viral replication. Given the abundance of HA on the viral surface, this protein is also the primary target for host innate and adaptive immune responses. Although addition of glycosylation sites on HA are a part of viral evolution to evade the host immune responses, there are specific glycosylation sites that are conserved during most of the evolution of the virus.

Decoding the distribution of glycan receptors for human-adapted influenza A viruses in ferret respiratory tract.

Ferrets are widely used as animal models for studying influenza A viral pathogenesis and transmissibility. Human-adapted influenza A viruses primarily target the upper respiratory tract in humans (infection of the lower respiratory tract is observed less frequently), while in ferrets, upon intranasal inoculation both upper and lower respiratory tract are targeted. Viral tropism is governed by distribution of complex sialylated glycan receptors in various cells/tissues of the host that are specifically recognized by influenza A virus hemagglutinin (HA), a glycoprotein on viral surface.

Pathogenesis and transmission of swine origin A(H3N2)v influenza viruses in ferrets.

Recent isolation of a novel swine-origin influenza A H3N2 variant virus [A(H3N2)v] from humans in the United States has raised concern over the pandemic potential of these viruses. Here, we analyzed the virulence, transmissibility, and receptor-binding preference of four A(H3N2)v influenza viruses isolated from humans in 2009, 2010, and 2011. High titers of infectious virus were detected in nasal turbinates and nasal wash samples of A(H3N2)v-inoculated ferrets.

Effect of D222G mutation in the hemagglutinin protein on receptor binding, pathogenesis and transmissibility of the 2009 pandemic H1N1 influenza virus.

Influenza viruses isolated during the 2009 H1N1 pandemic generally lack known molecular determinants of virulence associated with previous pandemic and highly pathogenic avian influenza viruses. The frequency of the amino acid substitution D222G in the hemagglutinin (HA) of 2009 H1N1 viruses isolated from severe but not mild human cases represents the first molecular marker associated with enhanced disease. To assess the relative contribution of this substitution in virus pathogenesis, transmission, and tropism, we introduced D222G by reverse genetics in the wild-type HA of the 2009 H1N1 virus, A/California/04/09 (CA/04).

A single base-pair change in 2009 H1N1 hemagglutinin increases human receptor affinity and leads to efficient airborne viral transmission in ferrets.

The 2009 H1N1 influenza A virus continues to circulate among the human population as the predominant H1N1 subtype. Epidemiological studies and airborne transmission studies using the ferret model have shown that the transmission efficiency of 2009 H1N1 viruses is lower than that of previous seasonal strains and the 1918 pandemic H1N1 strain. We recently correlated this reduced transmission efficiency to the lower binding affinity of the 2009 H1N1 hemagglutinin (HA) to α2→6 sialylated glycan receptors (human receptors).

Hemagglutinin receptor binding avidity drives influenza A virus antigenic drift.

Rapid antigenic evolution in the influenza A virus hemagglutinin precludes effective vaccination with existing vaccines. To understand this phenomenon, we passaged virus in mice immunized with influenza vaccine. Neutralizing antibodies selected mutants with single-amino acid hemagglutinin substitutions that increased virus binding to cell surface glycan receptors.

Transmission and pathogenesis of swine-origin 2009 A(H1N1) influenza viruses in ferrets and mice.

Recent reports of mild to severe influenza-like illness in humans caused by a novel swine-origin 2009 A(H1N1) influenza virus underscore the need to better understand the pathogenesis and transmission of these viruses in mammals. In this study, selected 2009 A(H1N1) influenza isolates were assessed for their ability to cause disease in mice and ferrets and compared with a contemporary seasonal H1N1 virus for their ability to transmit to naïve ferrets through respiratory droplets. In contrast to seasonal influenza H1N1 virus, 2009 A(H1N1) influenza viruses caused increased morbidity, replicated to higher titers in lung tissue, and were recovered from the intestinal tract of intranasally inoculated ferrets.