NA-Based mRNA Flu Vaccine Shows H1N1 Protection and Partial H5N1 Cross-Protection in Mice
An NA antigen to complement HA-focused flu vaccines
Influenza A viruses can change through antigenic drift and antigenic shift, reducing the effectiveness of existing vaccines. Researchers developed a messenger RNA (mRNA) vaccine candidate targeting neuraminidase (NA) on the viral surface and observed protection against H1N1 and limited cross-protection against H5N1 in mice. The study did not demonstrate preventive efficacy in people or complete prevention of infection.
Existing vaccines have mainly targeted hemagglutinin (HA), which helps the virus enter cells. When antigenic changes in HA cause a mismatch between a vaccine and circulating viruses, protection can decline. NA, by contrast, helps newly formed viruses exit cells and has a relatively more conserved sequence than HA, making it a candidate complementary antigen.
Animal study results for two computationally designed candidates
The researchers designed NA-D1 and NA-E2 using computational, phylogenetic, and structural analyses. They created three independent datasets by randomly sampling 704 N1 sequences per dataset from 4,849 human H1N1 N1 sequences from 2010–2023, then added sequences from three reference vaccine strains to each dataset. The 707 sequences reported in the paper refer to the size of each individual analysis dataset, not the total number of sequences collected. Unlike NA-D1, which reflects H1N1 characteristics, NA-E2 incorporated amino acid residues from H5N1 with the aim of broadening cross-recognition.
Mice aged 4–6 weeks received 10 μg of mRNA per antigen on days 0 and 14. The total dose in the bivalent vaccine group was 20 μg. On day 28, the mice were challenged with H1N1 or H5N1 virus; survival and body weight were monitored for 14 days, and lung viral loads were assessed on days 3 and 6 after infection.
After H1N1 challenge, all mice in the NA-D1-only group and the inactivated-vaccine group survived, recovering after temporary weight loss. The NA-E2-only group showed a similar pattern of protection, with somewhat greater variation. On day 6 after infection, infectious viral titers in the lungs were at the lowest levels in the NA-D1-only and bivalent groups. Because virus was detected in the lungs, these results should not be interpreted as complete prevention of infection.
For H5N1, survival was only about 40% in both the NA-E2-only and bivalent groups, and substantial weight loss occurred in all challenged groups. All mice in the inactivated-vaccine and buffer-control groups died. The bivalent group showed the most pronounced reduction in lung viral load on day 6 after infection. The virus used was A/chicken/Iksan/01/2006, described in the paper as a low-pathogenicity avian influenza virus. These findings do not support generalizing protection to recently circulating H5N1 viruses as a whole.
Potential for a universal vaccine and remaining validation needs
This study provides preclinical evidence that NA could serve as an antigen to complement HA-focused vaccines. However, sequence similarity does not necessarily mean functional inhibition by antibodies or broad protection. Indeed, some viruses showed low enzyme-inhibition activity despite high NA sequence similarity.
Combining HA and NA is a potential direction for further development, but these results do not establish that a universal flu vaccine has been achieved. Limitations include the mouse model, which does not reflect pre-existing immunity in people, and the absence of a comparator group receiving an H5N1 vaccine recommended by the World Health Organization (WHO). The study did not establish reductions in hospitalization or deaths in people, long-term protection, or a reduced need for annual vaccination.
The study design and results are available in the provided full paper.
Influenza A viruses continuously evolve through antigenic drift and shift, reducing the effectiveness of vaccines that rely primarily on hemagglutinin (HA). Neuraminidase (NA), a surface antigen with greater sequence conservation, has gained attention as a complementary target for broader influenza vaccine design. Using computational analyses of 707 post-2009 A(H1N1)pdm09 NA sequences, we designed two NA-based mRNA vaccine constructs: NA-D1, derived from contemporary H1N1 isolates, and NA-E2, incorporating conserved features shared between H1N1 and H5N1. Mice received prime-boost immunization followed by homologous H1N1 or heterologous H5N1 challenge. Both NA-D1 and NA-E2 induced NA-specific antibody titers and conferred complete protection against homologous H1N1 infection. In contrast, protection against heterologous H5N1 was partial, consistent with lower predicted antigenic similarity between the vaccine constructs and H5N1 NA. Together, these findings demonstrate that NA-based mRNA vaccination can elicit robust homologous protection but offers limited heterologous protection efficacy. Our results support NA as an important complementary antigen for next-generation influenza vaccines and highlight the potential of computationally guided, dual-antigen (HA + NA) strategies to advance the development of broadly protective mRNA vaccines.
For vaccine development, these findings can inform follow-up experiments that add NA as a complementary antigen to HA-focused candidates and compare survival, disease mitigation, and viral load. In particular, the reduction in lung viral load at the later time point after H5N1 challenge in the bivalent group merits further evaluation. However, the bivalent group received a higher total mRNA dose than the single-antigen groups, so the results cannot be attributed solely to the benefit of combining antigens. Further development should adjust antigen combinations and doses, include an appropriate H5N1 vaccine comparator, and assess the effects of pre-existing immunity. The current results do not provide grounds for changing vaccination recommendations or guaranteeing long-term protection in people.