Kaela Parkhouse

Potential antigenic explanation for atypical H1N1 infections among middle-aged adults during the 2013–2014 influenza season

Significance Influenza viruses typically cause a higher disease burden in children and the elderly, who have weaker immune systems. During the 2013–2014 influenza season, H1N1 viruses caused an unusually high level of disease in middle-aged adults. Here, we show that recent H1N1 strains possess a mutation that allows viruses to avoid immune responses elicited in middle-aged adults. We show that current vaccine strains elicit immune responses that are predicted to be less effective in some middle-aged adults. We suggest that new viral strains should be incorporated into seasonal influenza vaccines so that proper immunity is elicited in all humans, regardless of age and pre-exposure histories. Influenza viruses typically cause the most severe disease in children and elderly individuals. However, H1N1 viruses disproportionately affected middle-aged adults during the 2013–2014 influenza season. Although H1N1 viruses recently acquired several mutations in the hemagglutinin (HA) glycoprotein, classic serological tests used by surveillance laboratories indicate that these mutations do not change antigenic properties of the virus. Here, we show that one of these mutations is located in a region of HA targeted by antibodies elicited in many middle-aged adults. We find that over 42% of individuals born between 1965 and 1979 possess antibodies that recognize this region of HA. Our findings offer a possible antigenic explanation of why middle-aged adults were highly susceptible to H1N1 viruses during the 2013–2014 influenza season. Our data further suggest that a drifted H1N1 strain should be included in future influenza vaccines to potentially reduce morbidity and mortality in this age group.

Identification of Hemagglutinin Residues Responsible for H3N2 Antigenic Drift during the 2014–2015 Influenza Season

Influenza vaccines must be updated regularly because influenza viruses continuously acquire mutations in antibody binding sites of hemagglutinin (HA). The majority of H3N2 strains circulating in the Northern Hemisphere during the 2014-2015 season are antigenically mismatched to the A/Texas/50/2012 H3N2 vaccine strain. Recent H3N2 strains possess several new HA mutations, and it is unknown which of these mutations contribute to the 2014-2015 vaccine mismatch. Here, we use reverse genetics to demonstrate that mutations in HA antigenic site B are primarily responsible for the current mismatch. Sera isolated from vaccinated humans and infected ferrets and sheep had reduced hemagglutination inhibition and in vitro neutralization titers against reverse-genetics-derived viruses possessing mutations in the HA antigenic site B. These data provide an antigenic explanation for the low influenza vaccine efficacy observed during the 2014-2015 influenza season. Furthermore, our data support the World Health Organizations decision to update the H3N2 component of future vaccine formulations.

Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains

Significance The majority of influenza vaccine antigens are prepared in chicken eggs. Human vaccine strains grown in eggs often possess adaptive mutations that increase viral attachment to chicken cells. Most of these adaptive mutations are in the hemagglutinin protein, which functions as a viral attachment factor. Here, we identify a hemagglutinin mutation in the current egg-adapted H3N2 vaccine strain that alters antigenicity. We show that ferrets and humans exposed to the current egg-adapted H3N2 vaccine strain produce antibodies that poorly neutralize H3N2 viruses that circulated during the 2016–2017 influenza season. These studies highlight the challenges associated with producing influenza vaccine antigens in eggs, while offering a potential explanation of why there was only moderate vaccine effectiveness during the 2016–2017 influenza season. H3N2 viruses continuously acquire mutations in the hemagglutinin (HA) glycoprotein that abrogate binding of human antibodies. During the 2014–2015 influenza season, clade 3C.2a H3N2 viruses possessing a new predicted glycosylation site in antigenic site B of HA emerged, and these viruses remain prevalent today. The 2016–2017 seasonal influenza vaccine was updated to include a clade 3C.2a H3N2 strain; however, the egg-adapted version of this viral strain lacks the new putative glycosylation site. Here, we biochemically demonstrate that the HA antigenic site B of circulating clade 3C.2a viruses is glycosylated. We show that antibodies elicited in ferrets and humans exposed to the egg-adapted 2016–2017 H3N2 vaccine strain poorly neutralize a glycosylated clade 3C.2a H3N2 virus. Importantly, antibodies elicited in ferrets infected with the current circulating H3N2 viral strain (that possesses the glycosylation site) and humans vaccinated with baculovirus-expressed H3 antigens (that possess the glycosylation site motif) were able to efficiently recognize a glycosylated clade 3C.2a H3N2 virus. We propose that differences in glycosylation between H3N2 egg-adapted vaccines and circulating strains likely contributed to reduced vaccine effectiveness during the 2016–2017 influenza season. Furthermore, our data suggest that influenza virus antigens prepared via systems not reliant on egg adaptations are more likely to elicit protective antibody responses that are not affected by glycosylation of antigenic site B of H3N2 HA.

Successive annual influenza vaccination induces a recurrent oligoclonotypic memory response in circulating T follicular helper cells

Influenza vaccination induced a circulating TFH response involving influenza-specific memory T cell receptor clonotypes. Remembering TFH cell help Immunological memory operates on the assumption that if you’re exposed to an infection once, you’re more likely to be exposed again. Chance favors the prepared, and the memory response is bigger, faster, and stronger. Now, Herati et al. examine immunological memory in humans who have received successive annual influenza vaccinations. They find that circulating T follicular helper cells, which provide B cell help, not only respond to influenza vaccination but also form long-lasting memory. These cells may serve as markers for successful vaccination as well as targets for new vaccines. T follicular helper (TFH) CD4 cells are crucial providers of B cell help during adaptive immune responses. A circulating population of CD4 T cells, termed cTFH, have similarity to lymphoid TFH, can provide B cell help, and responded to influenza vaccination. However, it is unclear whether human vaccination-induced cTFH respond in an antigen-specific manner and whether they form long-lasting memory. We identified a cTFH population that expressed multiple T cell activation markers and could be readily identified by coexpression of inducible costimulator (ICOS) and CD38. This subset expressed more Bcl6, c-Maf, and interleukin-21 than did other blood CD4 subsets. Influenza vaccination induced a strong response in the ICOS+CD38+ cTFH at day 7, and this population included hemagglutinin-specific cells by tetramer staining and antigen-stimulated activation-induced marker expression. Moreover, T cell receptor β chain sequencing identified a clonal response in ICOS+CD38+ cTFH that strongly correlated with the increased cTFH frequency and was associated with the circulating plasmablast response. In participants who received successive annual vaccinations, a recurrent oligoclonal response was identified in the ICOS+CD38+ cTFH subset at 7 days after every vaccination. These oligoclonal responses in ICOS+CD38+ cTFH after vaccination persisted in the ICOS−CD38− cTFH repertoire in subsequent years, suggesting clonal maintenance in a memory reservoir in the more stable ICOS−CD38− cTFH subset. These data highlight the antigen specificity, lineage relationships, and memory properties of human cTFH responses to vaccination, providing new avenues for tracking and monitoring cTFH responses during infection and vaccination in humans.

Antibodies Against the Current Influenza A(H1N1) Vaccine Strain Do Not Protect Some Individuals From Infection With Contemporary Circulating Influenza A(H1N1) Virus Strains

Abstract During the 2013–2014 influenza season, nearly all circulating 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) strains possessed an antigenically important mutation in hemagglutinin (K166Q). Here, we performed hemagglutination-inhibition (HAI) assays, using sera collected from 382 individuals prior to the 2013–2014 season, and we determined whether HAI titers were associated with protection from A(H1N1)pdm09 infection. Protection was associated with HAI titers against an A(H1N1)pdm09 strain possessing the K166Q mutation but not with HAI titers against the current A(H1N1)pdm09 vaccine strain, which lacks this mutation. These data indicate that contemporary A(H1N1)pdm09 strains are antigenically distinct from the current A(H1N1)pdm09 vaccine strain.

Poor Immunogenicity, Not Vaccine Strain Egg Adaptation, May Explain the Low H3N2 Influenza Vaccine Effectiveness in 2012–2013

Abstract Background Influenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A(H3N2). Low VE has been attributed to mismatches between the vaccine and circulating influenza strains and to the vaccine’s elicitation of protective immunity in only a subset of the population. The low H3N2 VE in the 2012–2013 season was attributed to egg-adaptive mutations that created antigenic mismatch between the actual vaccine strain (IVR-165) and both the intended vaccine strain (A/Victoria/361/2011) and the predominant circulating strains (clades 3C.2 and 3C.3). Methods We investigated the basis of low VE in 2012–2013 by determining whether vaccinated and unvaccinated individuals were infected by different viral strains and by assessing the serologic responses to IVR-165, A/Victoria/361/2011, and 3C.2 and 3C.3 strains in an adult cohort before and after vaccination. Results We found no significant genetic differences between the strains that infected vaccinated and unvaccinated individuals. Vaccination increased titers to A/Victoria/361/2011 and 3C.2 and 3C.3 representative strains as much as to IVR-165. These results are consistent with the hypothesis that vaccination boosted cross-reactive immune responses instead of specific responses against unique vaccine epitopes. Only approximately one-third of the cohort achieved a ≥4-fold increase in titer. Conclusions In contrast to analyses based on ferret studies, low H3N2 VE in 2012–2013 in adults does not appear to be due to egg adaptation of the vaccine strain. Instead, low VE might have been caused by low vaccine immunogenicity in a subset of the population.

Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses.

T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4+ T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses.

Despite egg-adaptive mutations, the 2012-13 H3N2 influenza vaccine induced comparable antibody titers to the intended strain

Background Influenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A/H3N2. Failure to achieve consistently high VE has been attributed both to mismatches between the vaccine and circulating influenza strains and to the vaccines elicitation of protective immunity in only a subset of the population. The low H3N2 VE in 2012-13 was attributed to egg-adaptive mutations that created antigenic mismatch between the intended (A/Victoria/361/2011) and actual vaccine strain (IVR-165). Methods We investigate the basis of the low VE in 2012-2013 by evaluating whether vaccinated and unvaccinated individuals were infected by different viral strains and assessing the serologic responses to A/Victoria/361/2011 and the IVR-165 vaccine strain in an adult cohort before and after vaccination. Results We found no significant genetic differences between the strains that infected vaccinated and unvaccinated individuals. Vaccination increased titers to A/Victoria/361/2011 as much as to IVR-165. These results are consistent with the hypothesis that vaccination served merely to boost preexisting cross-reactive immune responses, which provided limited protection against infection with the circulating influenza strains. Conclusions In contrast to suggestive analyses based on ferret antisera, low H3N2 VE in 2012-13 does not appear to be due to the failure of the egg-adapted strain to induce a response to the intended vaccine strain. Instead, low VE might have been caused by the emergence of anti-genically novel influenza strains and low vaccine immunogenicity in a subset of the population.

Canine H3N8 influenza vaccines partially protect mice against the canine H3N2 strain currently circulating in the United States

Influenza A viruses infect many species and cross-species transmission occurs occasionally. An equine H3N8 influenza virus began circulating in dogs in 1999 and an avian H3N2 influenza virus began circulating in dogs in 2006. The canine H3N8 (cH3N8) viral strain has become endemic in parts of the United States and there is a commercially available vaccine against this strain. The canine H3N2 (cH3N2) strain did not circulate widely in the United States until 2015. Here, we used a mouse model to determine if the cH3N8 and cH3N2 strains are antigenically related and if a commercially available cH3N8 vaccine protects animals against the cH3N2 outbreak strain. We find that the cH3N8 vaccine elicits antibodies that react to internal viral proteins and the hemagglutinin stalk region of cH3N2 viruses. These antibodies do not provide sterilizing immunity against cH3N2 infection, but these antibodies limit cH3N2 replication in the lung.

The parasite-derived rOv-ASP-1 is an effective antigen-sparing CD4 + T cell-dependent adjuvant for the trivalent inactivated influenza vaccine, and functions in the absence of MyD88 pathway

Vaccination remains the most cost-effective biomedical approach for controlling influenza disease. In times of pandemics, however, these vaccines cannot be produced in sufficient quantities for worldwide use by the current manufacturing capacities and practices. What is needed is the development of adjuvanted vaccines capable of inducing an adequate or better immune response at a decreased antigen dose. Previously we showed that the protein adjuvant rOv-ASP-1 augments influenza-specific antibody titers and survival after virus challenge in both young adult and old-age mice when administered with the trivalent inactivated influenza vaccine (IIV3). In this study we show that a reduced amount of rOv-ASP-1, with 40-times less IIV3 can also induce protection. Apparently the potency of the rOv-ASP-1 adjuvanted IIV3 vaccine is independent of the IIV3-specific Th1/Th2 associated antibody responses, and independent of the presence of HAI antibodies. However, CD4+ T helper cells were indispensable for the protection. Further, rOv-ASP-1 with or without IIV3 elicited the increased level of various chemokines, which are known chemoattractant for immune cells, into the muscle 4 h after immunization, and significantly induced the recruitment of monocytes, macrophages and neutrophils into the muscles. The recruited monocytes had higher expression of the activation marker MHCII on their surface as well as CXCR3 and CCR2; receptors for IP-10 and MCP-1, respectively. These results show that the rOv-ASP-1 adjuvant allows substantial antigen sparing of IIV3 by stimulating at the site of injection the accumulation of chemokines and the recruitment of immune cells that can augment the activation of CD4+ T cell immune responses, essential for the production of antibody responses. Protection elicited by the rOv-ASP-1 adjuvanted IIV3 vaccine also appears to function in the absence of MyD88-signaling. Future studies will attempt to delineate the precise mechanisms by which the rOv-ASP-1 adjuvanted IIV3 vaccine works.