Novel influenza vaccine candidate shows promise

Clinical Trials & Research

While much of the media focus is currently on the coronavirus disease 2019 (COVID-19), the winterly influenza pandemic could be one of the deadliest in history, especially as COVID-19 cases continue to rise. As a result, researchers from the Icahn School of Medicine have been investigating the potential value of a new vaccine candidate.

Study: A Novel Recombinant Influenza Virus Neuraminidase Vaccine Candidate Stabilized by a Measles Virus Phosphoprotein Tetramerization Domain Provides Robust Protection from Virus Challenge in the Mouse Model. Image Credit: REDPIXEL.PL / Shutterstock.com

About the study

In the current study published in the journal mBio, researchers cloned sequences for the measles or Sendai virus phosphoprotein tetramerization domains/ a leucine zipper into the 5’ end of the sequence coding for the N1 Naq head domain of A/Michigan/45/15 to generate fusion proteins, which were expressed in insect cells via an N-terminal hexahistidine tag. This allowed the generation of constructs that could express high levels of recombinant tetrameric neuraminidase.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify the structural integrity of the recombinant proteins. To this end, the scientists observed that N1-MPP and N1-SEP showed the expected size bands for an N1 monomer head plus the tetramerization domain. Unfortunately, N1-SPP had an additional band suggesting degradation/cleavage of the protein.

When reducing agents were excluded, N1-SPP showed an additional band that suggested the formation of potentially dimeric structures. N1-MPP showed the same ability when a BS3 cross-linker was added; however, N1-SEP was unable to do so. As N1-SEP cannot form a stable tetramer, it was excluded from future experiments.

Enzyme-linked immunosorbent assays (ELISAs) were conducted using monoclonal antibodies IG01, 1000-ID05, and 4A5 to verify the correct display of antigenic epitopes. Mab binding to N1-MPP and N1-SPP showed a similar binding pattern to the positive control, N1-VASP. This suggests that the epitopes are presented in a native-like conformation.

An NA-Star assay revealed that both N1-MPP and N1-SPP exhibited high enzymatic activity, once again comparable to the positive control, thus indicating correct folding of the proteins. Pilot vaccination studies showed no significant weight loss in vivo.

N1-MPP was chosen to move forward in development, as it showed no degradation in the initial characterization. Unfortunately, this domain is deprived of the measles virus, and much of the population is vaccinated against measles. The researchers obtained sera from children pre- and post-measles vaccination and tested them against the N1-MPP through ELISA, which revealed very low reactivity post-vaccination.

The N1-MPP was expressed in Sf9 insect cells and purified, which was followed by the removal of the MPP tetramerization domain with thrombin to obtain the head. The crystal structure was resolved, showing a box-shaped tetramer that resembles other N1 structures. Further examination of the restudies revealed that the N1 NA head domain showed the correct conformation.

The scientists vaccinated mice in different groups, with N1-MPP either administered as a standalone vaccine (both adjuvanted and non-adjuvanted), as a supplement to seasonal quadrivalent inactivated influenza vaccine (QIV), or together with QIV but in the opposite leg of the mouse. There was a population of five mice per group. Mice were then challenged with QIV matched influenza A.

Vaccination with the N1-MPP construct protected the mice; however, these mice experienced 10% weight loss. Adjuvanted with AddaVax, the mice were fully protected, as were the other two experimental groups. All negative controls succumbed to the infection on days five and six.

The capacity of serum antibodies to bind to recombinant N1-VASp was tested to assess the characteristics of serum antibodies obtained post-transfection with N1-MPP. N1-VASP was used as the substrate to present antibodies that bind to the MPP tetramerization domain being detected.

After prime vaccination, strong responses were only detectable in the group that received the N1-MPP adjuvanted with AddaVax, although the N1-MPP and N1-MPP and QIV in separate legs did show weaker responses. Following the second vaccination, the antibody reactivities of these groups increased significantly, and the N1-MPP AddaVax NA inhibition increased dramatically.

When the N1-MPP was mixed with QIV, the response was lower as compared to when both shots were given in different legs. This finding has been supported in multiple previous studies, although it does not occur in every situation.

The researchers challenged six mice per group with a high dose of the H1N1 virus to determine whether the antibodies could inhibit the spread of the virus in lung tissue. The N1-MPP showed a high viral load that was only slightly better than the negative control group. However, the N1-MP AddaVax group showed a significantly lower titer, with three animals showing no viral load.

Conclusion

The authors highlight the value of their vaccine in helping to protect against influenza, not only against specific strains but against pandemic strains such as H2N2. While a suitable formulation still needs to be developed for transport and storage logistical purposes, the current study clearly indicates that N1-MPP can elicit a strong immune response and is a good candidate for further trials.

Journal reference:
  • Storhmeier, S., Amanat, F., Zhu, X., et al. (2021) A Novel Recombinant Influenza Virus Neuraminidase Vaccine Candidate Stabilized by a Measles Virus Phosphoprotein Tetramerization Domain Provides Robust Protection from Virus Challenge in the Mouse Model. mBio. https://journals.asm.org/doi/10.1128/mBio.02241-21

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