Development of Next-Generation COVID-19 Vaccines: BARDA Supported Phase 2b Study Designs.

In response to the COVID-19 pandemic, vaccines were quickly and successfully developed and deployed, saving millions of lives globally. While first generation vaccines are safe and effective in preventing disease caused by SARSCoV-2, next-generation vaccines have the potential to improve efficacy and safety. Vaccines delivered by a mucosal route may elicit greater protective immunity at respiratory surfaces thereby reducing transmission. Inclusion of viral antigens in addition to the spike protein may enhance protection against emerging variants of concern. Next-generation vaccine platforms with a new mechanism of action may necessitate efficacy trials to fulfill regulatory requirements. The Biomedical Advanced Research and Development Authority (BARDA) will be supporting Phase 2b clinical trials of candidate next-generation vaccines. The primary endpoint will be improved efficacy in terms of symptomatic disease relative to a currently approved COVID-19 vaccine. In this paper, we discuss the planned endpoints and potential challenges to this complex program.

Cross-Protection by Inactivated H5 Prepandemic Vaccine Seed Strains against Diverse Goose/Guangdong Lineage H5N1 Highly Pathogenic Avian Influenza Viruses.

The highly pathogenic avian influenza virus (HPAIV) H5N1 A/goose/Guangdong/1996 lineage (Gs/GD) is endemic in poultry across several countries in the world and has caused sporadic lethal infections in humans. Vaccines are important in HPAIV control both for poultry and in prepandemic preparedness for humans. This study assessed inactivated prepandemic vaccine strains in a One Health framework across human and agricultural and wildlife animal health, focusing on the genetic and antigenic diversity of field H5N1 Gs/GD viruses from the agricultural sector and assessing cross-protection in a chicken challenge model. Nearly half (47.92%) of the 48 combinations of vaccine and challenge viruses examined had bird protection of 80% or above. Most vaccinated groups had prolonged mean death times (MDT), and the virus-shedding titers were significantly lower than those of the sham-vaccinated group (P ≤ 0.05). The antibody titers in the prechallenge sera were not predictive of protection. Although vaccinated birds had higher titers of hemagglutination-inhibiting (HI) antibodies against the homologous vaccine antigen, most of them also had lower or no antibody titer against the challenge antigen. The comparison of all parameters and homologous or closely related vaccine and challenge viruses gave the best prediction of protection. Through additional analysis, we identified a pattern of epitope substitutions in the hemagglutinin (HA) of each challenge virus that impacted protection, regardless of the vaccine used. These changes were situated in the antigenic sites and/or reported epitopes associated with virus escape from antibody neutralization. As a result, this study highlights virus diversity, immune response complexity, and the importance of strain selection for vaccine development to control H5N1 HPAIV in the agricultural sector and for human prepandemic preparedness. We suggest that the engineering of specific antigenic sites can improve the immunogenicity of H5 vaccines.IMPORTANCE The sustained circulation of highly pathogenic avian influenza virus (HPAIV) H5N1 A/goose/Guangdong/1996 (Gs/GD) lineage in the agricultural sector and some wild birds has led to the evolution and selection of distinct viral lineages involved in escape from vaccine protection. Our results using inactivated vaccine candidates from the human pandemic preparedness program in a chicken challenge model identified critical antigenic conformational epitopes on H5 hemagglutinin (HA) from different clades that were associated with antibody recognition and escape. Even though other investigators have reported epitope mapping in the H5 HA, much of this information pertains to epitopes reactive to mouse antibodies. Our findings validate changes in antigenic epitopes of HA associated with virus escape from antibody neutralization in chickens, which has direct relevance to field protection and virus evolution. Therefore, knowledge of these immunodominant regions is essential to proactively develop diagnostic tests, improve surveillance platforms to monitor AIV outbreaks, and design more efficient and broad-spectrum agricultural and human prepandemic vaccines.

Mutations in the Neuraminidase-Like Protein of Bat Influenza H18N11 Virus Enhance Virus Replication in Mammalian Cells, Mice, and Ferrets.

To characterize bat influenza H18N11 virus, we propagated a reverse genetics-generated H18N11 virus in Madin-Darby canine kidney subclone II cells and detected two mammal-adapting mutations in the neuraminidase (NA)-like protein (NA-F144C and NA-T342A, N2 numbering) that increased the virus titers in three mammalian cell lines (i.e., Madin-Darby canine kidney, Madin-Darby canine kidney subclone II, and human lung adenocarcinoma [Calu-3] cells). In mice, wild-type H18N11 virus replicated only in the lungs of the infected animals, whereas the NA-T342A and NA-F144C/T342A mutant viruses were detected in the nasal turbinates, in addition to the lungs. Bat influenza viruses have not been tested for their virulence or organ tropism in ferrets. We detected wild-type and single mutant viruses each possessing NA-F144C or NA-T342A in the nasal turbinates of one or several infected ferrets, respectively. A mutant virus possessing both the NA-F144C and NA-T342A mutations was isolated from both the lung and the trachea, suggesting that it has a broader organ tropism than the wild-type virus. However, none of the H18N11 viruses caused symptoms in mice or ferrets. The NA-F144C/T342A double mutation did not substantially affect virion morphology or the release of virions from cells. Collectively, our data demonstrate that the propagation of bat influenza H18N11 virus in mammalian cells can result in mammal-adapting mutations that may increase the replicative ability and/or organ tropism of the virus; overall, however, these viruses did not replicate to high titers throughout the respiratory tract of mice and ferrets.IMPORTANCE Bats are reservoirs for several severe zoonotic pathogens. The genomes of influenza A viruses of the H17N10 and H18N11 subtypes have been identified in bats, but no live virus has been isolated. The characterization of artificially generated bat influenza H18N11 virus in mammalian cell lines and animal models revealed that this virus can acquire mammal-adapting mutations that may increase its zoonotic potential; however, the wild-type and mutant viruses did not replicate to high titers in all infected animals.

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice.

UNLABELLED: Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE: Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.

Hemagglutinin homologue from H17N10 bat influenza virus exhibits divergent receptor-binding and pH-dependent fusion activities.

Bat influenza virus H17N10 represents a distinct lineage of influenza A viruses with gene segments coding for proteins that are homologs of the surface antigens, hemagglutinin (HA) and neuraminidase (NA). Our recent study of the N10 NA homolog revealed an NA-like structure, but with a highly divergent putative active site exhibiting little or no NA activity, and provided strong motivation for performing equivalent structural and functional analyses of the H17 HA protein. The overall structure of the H17 HA homolog from A/little yellow-shouldered bat/Guatemala/060/2010 at 3.18 Å resolution is very similar to other influenza HAs, with a putative receptor-binding site containing some conserved aromatic residues that form the base of the sialic acid binding site. However, the rest of the H17 receptor-binding site differs substantially from the other HA subtypes, including substitution of other conserved residues associated with receptor binding. Significantly, electrostatic potential analyses reveal that this putative receptor-binding site is highly acidic, making it unfavorable to bind any negatively charged sialylated receptors, consistent with the recombinant H17 protein exhibiting no detectable binding to sialylated glycans. Furthermore, the fusion mechanism is also distinct; trypsin digestion with recombinant H17 protein, when exposed to pH 4.0, did not degrade the HA1 and HA2, in contrast to other HAs. These distinct structural features and functional differences suggest that the H17 HA behaves very differently compared with other influenza HAs.

Timing of influenza A(H5N1) in poultry and humans and seasonal influenza activity worldwide, 2004-2013.

Co-circulation of influenza A(H5N1) and seasonal influenza viruses among humans and animals could lead to co-infections, reassortment, and emergence of novel viruses with pandemic potential. We assessed the timing of subtype H5N1 outbreaks among poultry, human H5N1 cases, and human seasonal influenza in 8 countries that reported 97% of all human H5N1 cases and 90% of all poultry H5N1 outbreaks. In these countries, most outbreaks among poultry (7,001/11,331, 62%) and half of human cases (313/625, 50%) occurred during January-March. Human H5N1 cases occurred in 167 (45%) of 372 months during which outbreaks among poultry occurred, compared with 59 (10%) of 574 months that had no outbreaks among poultry. Human H5N1 cases also occurred in 59 (22%) of 267 months during seasonal influenza periods. To reduce risk for co-infection, surveillance and control of H5N1 should be enhanced during January-March, when H5N1 outbreaks typically occur and overlap with seasonal influenza virus circulation.

Detecting Spread of Avian Influenza A(H7N9) Virus Beyond China.

During February 2013-March 2015, a total of 602 human cases of low pathogenic avian influenza A(H7N9) were reported; no autochthonous cases were reported outside mainland China. In contrast, since highly pathogenic avian influenza A(H5N1) reemerged during 2003 in China, 784 human cases in 16 countries and poultry outbreaks in 53 countries have been reported. Whether the absence of reported A(H7N9) outside mainland China represents lack of spread or lack of detection remains unclear. We compared epidemiologic and virologic features of A(H5N1) and A(H7N9) and used human and animal influenza surveillance data collected during April 2013-May 2014 from 4 Southeast Asia countries to assess the likelihood that A(H7N9) would have gone undetected during 2014. Surveillance in Vietnam and Cambodia detected human A(H5N1) cases; no A(H7N9) cases were detected in humans or poultry in Southeast Asia. Although we cannot rule out the possible spread of A(H7N9), substantial spread causing severe disease in humans is unlikely.

Structural stability of influenza A(H1N1)pdm09 virus hemagglutinins.

UNLABELLED: The noncovalent interactions that mediate trimerization of the influenza hemagglutinin (HA) are important determinants of its biological activities. Recent studies have demonstrated that mutations in the HA trimer interface affect the thermal and pH sensitivities of HA, suggesting a possible impact on vaccine stability (). We used size exclusion chromatography analysis of recombinant HA ectodomain to compare the differences among recombinant trimeric HA proteins from early 2009 pandemic H1N1 viruses, which dissociate to monomers, with those of more recent virus HAs that can be expressed as trimers. We analyzed differences among the HA sequences and identified intermolecular interactions mediated by the residue at position 374 (HA0 numbering) of the HA2 subdomain as critical for HA trimer stability. Crystallographic analyses of HA from the recent H1N1 virus A/Washington/5/2011 highlight the structural basis for this observed phenotype. It remains to be seen whether more recent viruses with this mutation will yield more stable vaccines in the future. IMPORTANCE: Hemagglutinins from the early 2009 H1N1 pandemic viruses are unable to maintain a trimeric complex when expressed in a recombinant system. However, HAs from 2010 and 2011 strains are more stable, and our work highlights that the improvement in stability can be attributed to an E374K substitution in the HA2 subunit of the stalk that emerged naturally in the circulating viruses.

Identification of molecular markers associated with alteration of receptor-binding specificity in a novel genotype of highly pathogenic avian influenza A(H5N1) viruses detected in Cambodia in 2013.

Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with the alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to the absence of the mutations in poultry/environmental samples, suggested that the mutations occurred during human infection and did not transmit further.

New world bats harbor diverse influenza A viruses.

Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.

Antigenic analyses of highly pathogenic avian influenza a viruses.

In response to the ongoing threat to animal and human health posed by HPAI endemic in poultry, Asia (H5N1) and North America (H7N3) have revived efforts to reduce pandemic risk by disease control at the source and improved pandemic vaccines. Discovery of conserved neutralization epitopes in the HA, which mediate broad protection within and across HA subtypes have changed the paradigm of "broadly reactive" or "universal" vaccine design. Development of such vaccines would benefit from comparative antigenic analysis of viruses with increasing divergence within (and between) HA subtypes. A review of recent work to define the antigenic properties of HPAI viruses revealed data generated through an array of experimental approaches. This information has supported diagnostics and vaccine development for animal and human health. Further harmonization of analytical methods is needed to determine the antigenic relationships among multiple lineages of rapidly evolving HPAI viruses.

Crystal structures of two subtype N10 neuraminidase-like proteins from bat influenza A viruses reveal a diverged putative active site.

Recently, we reported a unique influenza A virus subtype H17N10 from little yellow-shouldered bats. Its neuraminidase (NA) gene encodes a protein that appears to be highly divergent from all known influenza NAs and was assigned as a new subtype N10. To provide structural and functional insights on the bat H17N10 virus, X-ray structures were determined for N10 NA proteins from influenza A viruses A/little yellow-shouldered bat/Guatemala/164/2009 (GU09-164) in two crystal forms at 1.95 Å and 2.5 Å resolution and A/little yellow-shouldered bat/Guatemala/060/2010 (GU10-060) at 2.0 Å. The overall N10 structures are similar to each other and to other known influenza NA structures, with a single highly conserved calcium binding site in each monomer. However, the region corresponding to the highly conserved active site of influenza A N1-N9 NA subtypes and influenza B NA differs substantially. In particular, most of the amino acid residues required for NA activity are substituted, and the putative active site is much wider because of displacement of the 150-loop and 430-loop. These structural features and the fact that the recombinant N10 protein exhibits no, or extremely low, NA activity suggest that it may have a different function than the NA proteins of other influenza viruses. Accordingly, we propose that the N10 protein be termed an NA-like protein until its function is elucidated.

A distinct lineage of influenza A virus from bats.

Influenza A virus reservoirs in animals have provided novel genetic elements leading to the emergence of global pandemics in humans. Most influenza A viruses circulate in waterfowl, but those that infect mammalian hosts are thought to pose the greatest risk for zoonotic spread to humans and the generation of pandemic or panzootic viruses. We have identified an influenza A virus from little yellow-shouldered bats captured at two locations in Guatemala. It is significantly divergent from known influenza A viruses. The HA of the bat virus was estimated to have diverged at roughly the same time as the known subtypes of HA and was designated as H17. The neuraminidase (NA) gene is highly divergent from all known influenza NAs, and the internal genes from the bat virus diverged from those of known influenza A viruses before the estimated divergence of the known influenza A internal gene lineages. Attempts to propagate this virus in cell cultures and chicken embryos were unsuccessful, suggesting distinct requirements compared with known influenza viruses. Despite its divergence from known influenza A viruses, the bat virus is compatible for genetic exchange with human influenza viruses in human cells, suggesting the potential capability for reassortment and contributions to new pandemic or panzootic influenza A viruses.

Genetic characterization of clade 2.3.2.1 avian influenza A(H5N1) viruses, Indonesia, 2012.

After reports of unusually high mortality rates among ducks on farms in Java Island, Indonesia, in September 2012, influenza A(H5N1) viruses were detected and characterized. Sequence analyses revealed all genes clustered with contemporary clade 2.3.2.1 viruses, rather than enzootic clade 2.1.3 viruses, indicating the introduction of an exotic H5N1 clade into Indonesia.

The 2009 pandemic influenza virus: where did it come from, where is it now, and where is it going?

Around 2008 or 2009, an influenza A virus that had been circulating undetected in swine entered human population. Unlike most swine influenza infections of humans, this virus established sustained human-to-human transmission, leading to a global pandemic. The virus responsible, 2009 pandemic H1N1 (H1N1pdm), is the result of multiple reassortment events that brought together genomic segments from classical H1N1 swine influenza virus, human seasonal H3N2 influenza virus, North American avian influenza virus, and Eurasian avian-origin swine influenza viruses. Genetically, H1N1pdm possesses a number of unusual features, although the genomic characteristics that permitted sustained human-to-human transmission are yet unclear. Human infection with H1N1pdm has generally resulted in low mortality, although certain subgroups (including pregnant women, people with some chronic medical conditions, morbidly obese individuals, and immunosuppressed people) have significantly higher risk of severe disease. As H1N1pdm has spread throughout the human population it continued to evolve. It has also reentered the swine population as a circulating pathogen, and has been transiently identified in other species such as turkeys, cats, and domestic ferrets. Most genetic changes in H1N1pdm to date have not been clearly linked to changes in antigenicity, disease severity, antiviral drug resistance, or transmission efficiency. However, the rapid evolution rate characteristic of influenza viruses suggests that changes in antigenicity are inevitable in future years. Experience with this first pandemic of twenty-first century reemphasizes the importance of influenza surveillance in animals as well as humans, and offers lessons to develop and enhance our ability to identify potentially pandemic influenza viruses in the future.

Investigating a crow die-off in January-February 2011 during the introduction of a new clade of highly pathogenic avian influenza virus H5N1 into Bangladesh.

We investigated unusual crow mortality in Bangladesh during January-February 2011 at two sites. Crows of two species, Corvus splendens and C. macrorhynchos, were found sick and dead during the outbreaks. In selected crow roosts, morbidity was ~1 % and mortality was ~4 % during the investigation. Highly pathogenic avian influenza virus H5N1 clade 2.3.2.1 was isolated from dead crows. All isolates were closely related to A/duck/India/02CA10/2011 (H5N1) with 99.8 % and A/crow/Bangladesh/11rs1984-15/2011 (H5N1) virus with 99 % nucleotide sequence identity in their HA genes. The phylogenetic cluster of Bangladesh viruses suggested a common ancestor with viruses found in poultry from India, Myanmar and Nepal. Histopathological changes and immunohistochemistry staining in brain, pancreas, liver, heart, kidney, bursa of Fabricius, rectum, and cloaca were consistent with influenza virus infection. Through our limited investigation in domesticated birds near the crow roosts, we did not identify any samples that tested positive for influenza virus A/H5N1. However, environmental samples collected from live-bird markets near an outbreak site during the month of the outbreaks tested very weakly positive for influenza virus A/H5N1 in clade 2.3.2.1-specific rRT-PCR. Continuation of surveillance in wild and domestic birds may identify evolution of new avian influenza virus and associated public-health risks.

Microevolution of highly pathogenic avian influenza A(H5N1) viruses isolated from humans, Egypt, 2007-2011.

We analyzed highly pathogenic avian influenza A(H5N1) viruses isolated from humans infected in Egypt during 2007-2011. All analyzed viruses evolved from the lineage of subtype H5N1 viruses introduced into Egypt in 2006; we found minimal evidence of reassortment and no exotic introductions. The hemagglutinin genes of the viruses from 2011 formed a monophyletic group within clade 2.2.1 that also included human viruses from 2009 and 2010 and contemporary viruses from poultry; this finding is consistent with zoonotic transmission. Although molecular markers suggestive of decreased susceptibility to antiviral drugs were detected sporadically in the neuraminidase and matrix 2 proteins, functional neuraminidase inhibition assays did not identify resistant viruses. No other mutations suggesting a change in the threat to public health were detected in the viral proteomes. However, a comparison of representative subtype H5N1 viruses from 2011 with older subtype H5N1 viruses from Egypt revealed substantial antigenic drift.

Structure and receptor complexes of the hemagglutinin from a highly pathogenic H7N7 influenza virus.

Recurrence of highly pathogenic avian influenza (HPAI) virus subtype H7 in poultry continues to be a public health concern. In 2003, an HPAI H7N7 outbreak in The Netherlands infected 89 people in close contact with affected poultry and resulted in one fatal case. In previous studies, the virus isolated from this fatal case, A/Netherlands/219/2003 (NL219) caused a lethal infection in mouse models and had increased replication efficiency and a broader tissue distribution than nonlethal isolates from the same outbreak. A mutation which introduces a potential glycosylation site at Asn123 in the NL219 hemagglutinin was postulated to contribute to the pathogenic properties of this virus. To study this further, we have expressed the NL219 hemagglutinin in a baculovirus expression system and performed a structural analysis of the hemagglutinin in complex with avian and human receptor analogs. Glycan microarray and kinetic analysis were performed to compare the receptor binding profile of the wild-type recombinant NL219 HA to a variant with a threonine-to-alanine mutation at position 125, resulting in loss of the glycosylation site at Asn123. The results suggest that the additional glycosylation sequon increases binding affinity to avian-type α2-3-linked sialosides rather than switching to a human-like receptor specificity and highlight the mechanistic diversity of these pathogens, which calls attention to the need for further studies to fully understand the unique properties of these viruses.

Adaptation of a duck influenza A virus in quail.

Quail are thought to serve as intermediate hosts of influenza A viruses between aquatic birds and terrestrial birds, such as chickens, due to their high susceptibility to aquatic-bird viruses, which then adapt to replicate efficiently in their new hosts. However, does replication of aquatic-bird influenza viruses in quail similarly result in their efficient replication in humans? Using sialic acid-galactose linkage-specific lectins, we found both avian (sialic acid-α2-3-galactose [Siaα2-3Gal] linkages on sialyloligosaccharides)--and human (Siaα2-6Gal)-type receptors on the tracheal cells of quail, consistent with previous reports. We also passaged a duck H3N2 virus in quail 19 times. Sequence analysis revealed that eight mutations accumulated in hemagglutinin (HA) during these passages. Interestingly, many of the altered HA amino acids found in the adapted virus are present in human seasonal viruses, but not in duck viruses. We also found that stepwise stalk deletion of neuraminidase occurred during passages, resulting in reduced neuraminidase function. Despite some hemagglutinin mutations near the receptor binding pocket, appreciable changes in receptor specificity were not detected. However, reverse-genetics-generated viruses that possessed the hemagglutinin and neuraminidase of the quail-passaged virus replicated significantly better than the virus possessing the parent HA and neuraminidase in normal human bronchial epithelial cells, whereas no significant difference in replication between the two viruses was observed in duck cells. Further, the quail-passaged but not the original duck virus replicated in human bronchial epithelial cells. These data indicate that quail can serve as intermediate hosts for aquatic-bird influenza viruses to be transmitted to humans.