Altering the Immunogenicity of Hemagglutinin Immunogens by Hyperglycosylation and Disulfide Stabilization

Influenza virus alters glycosylation patterns on its surface exposed glycoproteins to evade host adaptive immune responses. The viral hemagglutinin (HA), in particular the H3 subtype, has increased its overall surface glycosylation since its introduction in 1968. We previously showed that modulating predicted N-linked glycosylation sites on H3 A/Hong Kong/1/1968 HA identified a conserved epitope at the HA interface. This epitope is occluded on the native HA trimer but is likely exposed during HA “breathing” on the virion surface. Antibodies directed to this site are protective via an ADCC-mediated mechanism. This glycan engineering strategy made an otherwise subdominant epitope dominant in the murine model. Here, we asked whether cysteine stabilization of the hyperglycosylated HA trimer could reverse this immunodominance by preventing access to the interface epitope and focus responses to the HA receptor binding site (RBS). While analysis of serum responses from immunized mice did not show a redirection to the RBS, cysteine stabilization did result in an overall reduction in immunogenicity of the interface epitope. Thus, glycan engineering and cysteine stabilization are two strategies that can be used together to alter immunodominance patterns to HA. These results add to rational immunogen design approaches used to manipulate immune responses for the development of next-generation influenza vaccines.

Rapid Differential Detection of H1 and H3 Subtype Swine Influenza Viruses by a TaqMan-MGB Based Duplex One-Step Real Time RT-PCR Assay

Swine influenza is not only an economically important respiratory disease in swine, but also constantly poses a threat to human health. Hence, developing a rapid, sensitive and efficient detection method of swine influenza virus (SIV) is highly essential. By aligning the HA gene sequences of SIV circulating in China in recent 10 years, a H1 primer-probe set targeting both Eurasian avian-like H1N1 (EA H1N1) and Pandemic 2009 H1N1 (Pdm09 H1N1) lineages plus a H3 prime-probe set targeting the prevalent human-like H3N2 (HL H3N2) subtype were designed, respectively. Further, a TaqMan-MGB based duplex one-step real time RT-PCR (RRT-PCR) assay was established and evaluated. The duplex RRT-PCR possessed the detection limit of 5 copies/μL HA plasmid for each of the EA H1N1, Pdm09 H1N1 and HL H3N2 subtype SIVs, and matched an overall detection sensitivity of 100% and specificity of 91.67% with traditional virus isolation through chicken embryo inoculation using experimentally infected mice lung samples. Besides, the method showed high repeatability both within-run and between-runs, and no cross-reactivity against some commonly circulated porcine viruses in China. Furthermore, the duplex RRT-PCR method revealed a relatively higher prevalent rate of H1 than H3 subtype SIV in 166 nasal swabs from pigs in some slaughterhouse during October ~ December, 2019. This developed assay could be very helpful for rapid differential detection and routine surveillance of EA H1N1, Pdm09 H1N1 and HL H3N2 subtype SIVs in China.

Gaps in Serologic Immunity against Contemporary Swine-Origin Influenza A Viruses among Healthy Individuals in the United States

Influenza A Viruses (IAV) in domestic swine (IAV-S) are associated with sporadic zoonotic transmission at the human–animal interface. Previous pandemic IAVs originated from animals, which emphasizes the importance of characterizing human immunity against the increasingly diverse IAV-S. We analyzed serum samples from healthy human donors (n = 153) using hemagglutination-inhibition (HAI) assay to assess existing serologic protection against a panel of contemporary IAV-S isolated from swine in the United States (n = 11). Age-specific seroprotection rates (SPR), which are the proportion of individuals with HAI ≥ 1:40, corresponded with lower or moderate pandemic risk classifications for the multiple IAV-S examined (one H1-δ1, one H1-δ2, three H3-IVA, one H3-IVB, one H3-IVF). Individuals born between 2004 and 2013 had SPRs of 0% for the five classified H3 subtype IAV-S, indicating youth may be particularly predisposed to infection with these viruses. Expansion of existing immunologic gaps over time could increase likelihood of future IAV-S spillover to humans and facilitate subsequent sustained human-to-human transmission resulting in disease outbreaks with pandemic potential.

Novel Reassortant H3N2 Avian Influenza Virus Isolated from Domestic Ducks in Eastern China in 2016

ABSTRACT H3 subtype avian influenza virus (AIV) poses a great threat to public health, and so investigating its epidemiology is of great importance. A novel reassortant H3N2 AIV strain was isolated from a live poultry market in eastern China. The strain’s genes originated from H1N1, H3, and H7 AIVs. Thus, the genome information of the H3N2 isolate will help to investigate further the epidemiology of H3 subtype AIVs in China.