There has been an increase in the development of coronavirus disease 2019 (COVID-19) vaccines in several countries due to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The RNA vaccine platform is a next-generation platform that comprises messenger RNA (mRNA), which helps to encode the target antigen. The first concept of an mRNA vaccine was developed in 1990; however, its application was limited due to ineffective in vivo delivery, instability, and high innate immunogenicity of mRNA.

Technological advances in lipid nanoparticles (LNPs), as well as the introduction of nucleoside modification by pseudouridine can help to overcome such limitations. The integration of mRNA does not take place into the host genome. This allows the manufacture of mRNA in a cell-free manner, resulting in cost-effective, scalable, and rapid production.

Authorization of two COVID-19 mRNA vaccines (Pfizer/BioNTech BNT162b2 and Moderna mRNA-1273) took place by the US Food and Drug Administration (FDA) for emergency use within a year of their development. Other COVID-19 vaccines, such as viral vector and inactivated vaccines, have also been approved and administered worldwide.

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Recently, a traditional protein subunit type vaccine, Novavax has been approved by the FDA for emergency use. The availability of these vaccines has led to the administration of different types of vaccines (heterologous prime-boost vaccination) to immunize people against a single virus.

Studies have reported heterologous prime-boost strategies to induce higher T-cell responses and neutralizing antibody titers. However, the efficacy and immunogenicity of heterologous priming-boosting using mRNA and protein vaccines are unknown. The impact of the immunization sequence on vaccine efficacy is also not reported.

The influenza virus is an important zoonotic virus that causes about 3 to 5 million severe illness cases and 290,000 to 650,000 deaths globally each year. Although current influenza vaccines are effective against the virus, the emergence of novel pandemic strains r failure to predict the vaccine strain can lead to a reduction in their effectiveness. Therefore, developing an influenza vaccine that can be rapidly produced is important.

Currently, a few quadrivalent and monovalent influenza vaccines that encode hemagglutinin (HA) from seasonal influenza strains are undergoing clinical trials, and many others are in the preclinical phase.

A new study available on as a preprint on Research Square* and under review at npj Vaccines aimed to analyze whether the order of immunization of vaccine types impacted the efficacy of a heterologous prime-boost vaccination strategy.

About the study

The study involved six-week-old female BALB/c mice acclimatized for one week before beginning the experiment. The DNA template for the mRNA vaccine was obtained from a DNA fragment that encoded the HA protein of the influenza A virus, followed by the synthesis of mRNA-HA. Thereafter, transfection of Vero cells took place using 10 µg mRNA followed by western blot.

Formulation of LNPs took place along with their characterization. Immunization of mice took place at a 2-week interval using 5 µg mRNA-HA or 1 µg HA protein. Elisa was used to determine antibody levels, followed by an ELISpot assay.

Mice were then challenged with the virus, after which clinical illness, survival, and body weight were assessed. Real-time polymerase chain reaction (PCR) was carried out using total RNA from bronchoalveolar lavage fluid (BALF) and lungs. Finally, BALF collection and histopathological analysis were done.

Study findings

The results indicated that priming with led to high levels of IgG2a, whereas priming with protein-HA led to an IgG1-biased response. Homologous mRNA-HA-immunization (R-R) and heterologous mRNA-HA/protein-HA-immunization (R-P) were observed to lead to balanced IgG1/IgG2a responses.

The R-P group was observed to show a higher hemagglutination inhibition (HI) and microneutralization (MN) as compared to the P-R group. No significant differences regarding the interferon-γ (IFN-γ) cytokine-producing cells in splenocytes were observed between the R-P and P-R groups.

A higher frequency of antigen-specific IFN-γ producing cells in CD4 + T cells was observed in the R-P group as compared to the P-P and P-R groups. A higher frequency of IFN-γ or TNF-α producing cells in CD8 + T cells was also observed in the R-R group, while a higher frequency of interleukin-2 (IL-2)-producing cells in CD4 + T cells was observed in P-R and R-P groups. Moreover, higher numbers of CD4 + and CD8 + T cells were reported in the R-P and P-R groups as compared to the P-P group.

Different gene expression patterns were observed in the P-P and R-R groups but not between R-P and P-R groups. The P-P and P-R groups were observed to show increased neutrophil degranulation and mast cell pathways. Moreover, increased stimulatory C-type lectin receptor signaling pathways, cytotoxic T cell differentiation pathways, and helper T cell diapedesis were also increased in the P-R group.

The R-P group was observed to have similar enriched pathways along with increased CD8 + T-cell activation and Th2 differentiation pathways. The R-R group was observed to show increased regulation of the dendritic cell pathway, innate immune response signaling, Th2, and cytotoxic T-cell differentiation pathways.

Additionally, it was also observed to show increased expression of Bcl6, which is a transcription factor for Hmgb1 and follicular helper T-cells, along with interferon regulatory factor 1 (Irf1) and V-set immunoregulatory receptor.

Furthermore, no significant difference was observed concerning the protective efficacy of the homologous and heterologous prime-boost regimes. Mild to moderate lung changes were observed in the P-R group, while minimal to mild changes were observed in the R-P group. The viral titers in the BALF and lungs were observed to be reduced 1-week post-viral challenge in the R-P group compared to the P-R group. IgG2a levels were observed to be higher in the P-R group as compared to the R-P group.

Higher percentages of IL-2, TNF-α, and IFN-γ producing CD4 +T cells were observed in the R-P group, while higher percentages of IL-2, TNF-α, and IFN-γ producing CD8 + T cells were observed in the P-R group. Additionally, the central CD8 + T cells as well as proliferating effector CD4 + and CD8 + T cells following viral challenge, were observed to be lowest in the R-P group.


Therefore, the current study suggests that a heterologous vaccination strategy with an initial inoculation with an mRNA vaccine followed by a secondary or tertiary inoculation with a protein vaccine might be the most effective and safe vaccination strategy against the virus.

*Important notice

Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

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