A long-term stable cold-chain-friendly HIV mRNA vaccine encoding multi-epitope viral protease cleavage site immunogens inducing immunogen-specific protective T cell immunity

Abstract

The lack of success in clinical trials for HIV vaccines highlights the need to explore novel strategies for vaccine development. Research on highly exposed seronegative (HESN) HIV-resistant Kenyan female sex workers revealed naturally protective immunity is correlated with a focused immune response mediated by virus-specific CD8 T cells. Further studies indicated that the immune response is unconventionally focused on highly conserved sequences around HIV viral protease cleavage sites (VPCS). Thus, taking an unconventional approach to HIV vaccine development, we designed lipid nanoparticles loaded with mRNA that encodes multi-epitopes of VPCS (MEVPCS-mRNA LNP), a strategic design to boost antigen presentation by dendritic cells, promoting effective cellular immunity. Furthermore, we developed a novel cold-chain compatible mRNA LNP formulation, ensuring long-term stability and compatibility with cold-chain storage/transport, widening accessibility of mRNA LNP vaccine in low-income countries. The in-vivo mouse study demonstrated that the vaccinated group generated VPCS-specific CD8 memory T cells, both systemically and at mucosal sites of viral entry. The MEVPCS-mRNA LNP vaccine-induced CD8 T cell immunity closely resembled that of the HESN group and displayed a polyfunctional profile. Notably, it induced minimal to no activation of CD4 T cells. This proof-of-concept study underscores the potential of the MEVPCS-mRNA LNP vaccine in eliciting CD8 T cell memory specific to the highly conserved multiple VPCS, consequently having a broad coverage in human populations and limiting viral escape mutation. The MEVPCS-mRNA LNP vaccine holds promise as a candidate for an effective prophylactic HIV vaccine.

Keywords: HIV vaccine; cold-chain friendly mRNA LNPs; multi-epitope viral PCS; prophylaxis; protective immunity.

Figure 1.

MEVPCS-mRNA synthesis and nanoencapsulation strategy. (A) The 12 VPCS sequences of Gag, Gag-Pol, and Nef precursor proteins from SIVmac239 strain. (B) The MEVPCS polypeptide sequence with a GGS spacers. (C) The codon-optimized MEVPCS-mRNA sequence as expressed from MEVPCS_GGS pBluescript II KS(+) clone. (D) Schematic diagram depicting MEVPCS-mRNA LNP synthesis method. (E) TEM image of MEVPCS-mRNA LNPs.

Figure 2.

MEVPCS-mRNA LNP stability study over 2 months at 4–8°C. The stability was analysed based on three variables, i.e. size (nm), concentration (LNP/mL), and surface charge (ζ-potential, mV). Each variable shows the mean ± standard deviation of means (SEM) of six MEVPCS-mRNA LNP batches. Each data point in the graph represents data from one MEVPCS-mRNA LNP batch. Statistical analysis (two-way ANOVA with post-Šídák’s multiple comparisons test) showed no significant differences between 0-, 30-, and 60-day study time in all tested variables.

Figure 3.

MEVPCS polypeptide expression study based on WB analysis. (A) In-vitro translation (IVT) of MEVPCS polypeptide (15 µL IVT product/well); Lane 2, “*” and Lane 3, “#” indicates MEVPCS expression from MEVPCS-mRNA and linearized MEVPCS_GGS (pBluescript plasmid, respectively) using 1-Step Human Coupled IVT Kit. The anti-Vinculin antibody (MW124 kDa), was used as a higher MW housekeeping protein. (B) Represents in-vitro expression of MEVPCS polypeptide in HEK293T cells at different concentrations and different time points along with anti-β-actin (MW: 46 kDa), as lower MW housekeeping protein. (C) Comparative graphical presentation of MEVPCS polypeptide expression based on band-intensity analysis. Each data point represents the mean of four independent experiments.

Figure 4.

MEVPCS-mRNA LNP in-vivo vaccine potency study. (A) Schematic diagram depicting the in-vivo study strategy (A). Graphical presentation showing the change in CD4+ population (B) and CD8+ population (C) subpopulation phenotype at two weeks post-prime and -boost immunization (day 28). The study was conducted on PBMCs isolated from whole blood collected from mice on the terminal day (day 28). The graph presents data as mean ± SD, and each data obtained was presented (solid dot: MEVPCS-mRNA LNP group; hollow dot: LNP (blank) group; and cross: untreated control (PBS) group). The MEVPCS-mRNA LNP group had eight mice, whereas, the LNP group and the untreated Control (PBS) had six mice each. The statistical difference between groups was analysed by one-way ANOVA followed by Dunnett’s multiple comparisons.

Figure 5.

VPCS-specific immune response evaluation study. The graph presented the comparative immunophenotypic difference observed in CD8+ T-lymphocytes upon 18 h VPCS stimulation of PBMCs (A), single cells from draining lymph nodes (B), spleen (C), cervicovaginal tissue (D), and small intestine (E), isolated from respective tissues collected from two weeks post-prime and -boost immunization mice (day 28). The statistical difference was analysed by two-way ANOVA followed by Bonferroni’s multiple comparisons test (post-test). The graph presents all data obtained, with a median line. Data presented were obtained from eight mice for the MEVPCS-mRNA LNP group and six mice for the untreated Control (PBS). Each data point from the respective group was presented by their respective symbols (solid circle: MEVPCS-mRNA LNP group; hollow downward triangle: untreated control (PBS group)). The asterisk mark “*,” “**,” and “****” represent p-value “<0.05,” “<0.01,” and “<0.0001,” respectively.

Figure 6.

Comparative cytokine and chemokine profile of supernatant from VPCS-stimulated PBMCs. (A) Heatmap showing net fold change (relative to the untreated control group) in 40 tested cytokine/chemokine levels in MEVPCS-mRNA LNP group vs. LNP group. Graph demonstrating the relative level of secreted pro-inflammatory (B) and anti-inflammatory (C) cytokines/chemokines. The heat map data represents a geographical mean (A) whereas the graphical data (B–C) are presented as mean ± SD obtained from MEVPCS-mRNA LNP group (n = 6), LNP group (n = 3), and untreated Control (PBS) (n = 3). The two-way ANOVA statistical analysis followed by the post-test Bonferroni’s multiple comparisons test was used to predict the significant change. The asterisk marks “*” and “**” represent p-value “<0.05” and “<0.01,” respectively.