Laura Lazo

The COVID-19 pandemic has had a profound impact on the world. While the disease is currently under control, the emergence of new coronavirus variants with pandemic potential underscores the need for more universal coronavirus vaccines. This study evaluates the immunogenicity in non-human primates of two broad-spectrum nasal vaccine candidates called PanCoV. The vaccine preparations are based on highly conserved regions of SARS-CoV-2, specifically the nucleocapsid (N) and the S2 subunit from Spike protein, which include several relevant T-cell epitopes and have the potential to form virus-like particles. PanCoV1 candidate contains the N protein, while the PanCoV2 comprises a chimeric protein containing the C-terminal domain of N protein fused to a fragment of S2 subunit. Both vaccine candidates also include the receptor-binding domain (RBD) from Spike protein and the ODN-39M as CpG adjuvant. The results demonstrate that both nasal vaccine candidates can boost the anti-RBD immune response and induce anti-N immunity, this systemic response shows cross-reactivity with SARS-CoV, MERS-CoV, and H-CoV antigens. In addition, a neutralizing response against SARS-CoV-2 that persisted for almost three months after the last dose was induced. A mucosal-specific and cross-reactive IgA response was also observed, mainly in monkeys vaccinated with PanCoV2. Furthermore, the specific IFNγ-secreting response observed in the vaccinated groups peripheral blood mononuclear cells highlights the ability of these candidates to stimulate cellular immunity with a Th1 bias. PanCoV vaccines emerge as promising candidates for use as a booster alternative, with the potential to amplify and broaden the scope of the immune response against coronaviruses.

Despite the rapid development of vaccines against COVID-19, they have important limitations, such as safety issues, the scope of their efficacy, and the induction of mucosal immunity. The present study proposes a potential component for a new generation of vaccines. The recombinant nucleocapsid (N) protein from the SARS-CoV-2 Delta variant was combined with the ODN-39M, a synthetic 39 mer unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), used as an adjuvant. The evaluation of its immunogenicity in Balb/C mice revealed that only administration by intranasal route induced a systemic cross-reactive, cell-mediated immunity (CMI). In turn, this combination was able to induce anti-N IgA in the lungs, which, along with the specific IgG in sera and CMI in the spleen, was cross-reactive against the nucleocapsid protein of SARS-CoV-1. Furthermore, the nasal administration of the N + ODN-39M preparation, combined with RBD Delta protein, enhanced the local and systemic immune response against RBD, with a neutralizing capacity. Results make the N + ODN-39M preparation a suitable component for a future intranasal vaccine with broader functionality against Sarbecoviruses.

Despite the rapid development of vaccines and their reported efficacy for controlling the COVID-19 waves, two key challenges remain: the scope of the immunity against upcoming variants and zoonosis events, and the induction of mucosal immunity able to clear the virus in the upper respiratory tract for halting the transmission. The present study is aiming at assessing a potential component for a new generation of vaccines so as to overcome such limitations. The recombinant nucleocapsid (N) protein from SARS-CoV-2 Delta variant was combined with a phosphodiester backbone CpG ODN (ODN-39M), forming high molecular weight aggregates. The evaluation of its immunogenicity in Balb/C mice revealed that only administration by intranasal route induced a systemic cross-reactive Cell-Mediated-Immunity (CMI). In turn, this combination was able to induce anti-N IgA in lungs, which along with the specific IgG in sera and CMI in spleen, resulted cross-reactive against the nucleocapsid protein of SARS-CoV-1. Furthermore, the nasal administration of the N+ODN-39M preparation combined with the RBD Delta protein, as inductor of neutralizing Abs, enhanced the local and systemic immune response against RBD with a modulation toward a Th1 pattern. Taken together, these results make the N+ODN-39M preparation a suitable component for a future intranasal pancorona vaccine against Sarbecoviruses. Particularly, the bivalent vaccine formulation N+ODN-39M+RBD could be used as an effective nasal booster in previously vaccinated population.

Introduction: The induction of a functional immune response against the four viral serotypes is one of the premises for an effective vaccine against Dengue virus. This is challenging since the immunization with four antigens leads to immunologic phenomena such as antigen interference, immuno-dominance and tolerance. Moreover, the four serotypes have intrinsic features which impact the outcome after the immunization with a tetravalent formulation. Areas covered: This work reviews the main studies evidencing the differences between Dengue virus 4 and the rest of the serotypes. We address some peculiarities of this virus and discuss which factors could explain the heterogeneous response achieved after the immune evaluation of tetravalent formulations. Expert Opinion: The low immunogenicity associated to serotype 4 could slow down the development of a vaccine against Dengue virus. Achieving similar levels of neutralizing antibodies against the four serotypes has been the goal of many vaccine developers. However, this does not need to be seen as a mandatory dogma. High levels of efficacy against Dengue virus 4 could be reached even if it shows the lowest neutralizing antibody titers among the viral complex. Studies on the efficacy of vaccines, currently in phase III clinical trials, should shed light on this concern in the near future.

There are several dengue vaccine candidates at advanced stages of development, but none of them are licensed. Despite the reactogenicity and immunogenicity profile in humans of the tetravalent ChimeriVax™ dengue vaccine candidate, in efficacy trials, it has failed to confer complete protection against dengue virus (DENV)-1 and DENV-2. However, full protection against the four serotypes had been observed previously in monkeys immunized with this vaccine candidate. Some authors have tried to explain this contradiction by hypothesizing that protection rates in non-human primates (NHPs) are associated with a lack of post-challenge anamnestic immune responses. Here, we studied the protection and anamnestic response patterns after homologous challenge in NHPs previously infected with DENV-2. Two immunization schemes were used, varying the viral doses and the intervals between them. Animals developed immunity against DENV-2 that provided full protection against reinfection with a homologous virus. However, all monkeys showed a significant increase in antiviral and neutralizing antibody titers after challenge. Our results suggest that sterilizing immunity could not be induced by infection with the virus despite the lack of detectable viremia in some animals in which an increase in antibody titer was observed. For this reason, we propose that the lack of an anamnestic neutralizing antibody response after challenge, as suggested by some authors, should be carefully reviewed as a criterion for evaluating the functionality of vaccine candidates.

Our group developed a subunit vaccine candidate against dengue virus based on two different viral regions: the domain III of the envelope protein and the capsid protein. The novel chimeric protein from dengue-2 virus [domain III-capsid (DIIIC-2)], when presented as aggregated incorporating oligodeoxynucleotides, induced anti-viral and neutralizing antibodies, a cellular immune response and conferred significant protection to mice and monkeys. The remaining constructs were already obtained and properly characterized. Based on this evidence, this work was aimed at assessing the immune response in mice of the chimeric proteins DIIIC of each serotype, as monovalent and tetravalent formulations. Here, we demonstrated the immunogenicity of each protein in terms of humoral and cell-mediated immunity, without antigen competition on the mixture forming the formulation tetra DIIIC. Accordingly, significant protection was afforded as measured by the limited viral load in the mouse encephalitis model. The assessment of the tetravalent formulation in non-human primates was also conducted. In this animal model, it was demonstrated that the formulation induced neutralizing antibodies and memory cell-mediated immune response with IFN-γ-secreting and cytotoxic capacity, regardless the route of immunization used. Taken together, we can assert that the tetravalent formulation of DIIIC proteins constitutes a promising vaccine candidate against dengue virus, and propose it for further efficacy experiments in monkeys or in the dengue human infection model, as it has been recently proposed.

Previously, we reported the ability of the chimeric protein DIIIC‐2 (domain III of the dengue envelope protein fused to the capsid protein of dengue‐2 virus), to induce immunity and protection in mice, when it is highly aggregated with a non‐defined oligodeoxynucleotide (ODN) and adjuvanted in alum. In this work, three different defined ODNs were studied as aggregating agents. Our results suggest that the nature of the ODN influences the capacity of protein DIIIC‐2 to activate cell‐mediated immunity in mice. Consequently, the ODN 39M was selected to perform further experiments in mice and nonhuman primates. Mice receiving the preparation 39M‐DIIIC‐2 were solidly protected against dengue virus (DENV) challenge. Moreover, monkeys immunized with the same preparation developed neutralizing antibodies, as measured by four different neutralization tests varying the virus strains and the cell lines used. Two of the immunized monkeys were completely protected against challenge, whereas the third animal had a single day of low‐titer viremia. This is the first work describing the induction of short‐term protection in monkeys by a formulation that is suitable for human use combining a recombinant protein from DENV with alum.