Evaluation of repRNA vaccine for induction and in utero transfer of maternal antibodies in a pregnant rabbit model

Abstract

Mother-to-child transmission is a major route for infections in newborns. Vaccination in mothers to leverage the maternal immune system is a promising approach to vertically transfer protective immunity. During infectious disease outbreaks, such as the 2016 Zika virus (ZIKV) outbreak, rapid availability of vaccines can prove critical in reducing widespread disease burden. The recent successes of mRNA vaccines support their evaluation in pregnant animal models to justify their use in neonatal settings. Here we evaluated immunogenicity of self-amplifying replicon (repRNA) vaccines, delivered with our clinical-stage LION nanoparticle formulation, in pregnant rabbits using ZIKV and HIV-1 as model disease targets. We showed that LION/repRNA vaccines induced robust antigen-specific antibody responses in adult pregnant rabbits that passively transferred to newborn kits in utero. Using a matrixed study design, we further elucidate the effect of vaccination in kits on the presence of pre-existing maternal antibodies. Our findings showed that timing of maternal vaccination is critical in maximizing in utero antibody transfer, and subsequent vaccination in newborns maintained elevated antibody levels compared with no vaccination. Overall, our results support further development of the LION/repRNA vaccine platform for maternal and neonatal settings.

Keywords: HIV-1; LION formulation; ZIKV; humoral immunity; maternal transfer; mother-to-child transmission; nanoparticle emulsion; pregnant rabbit model; self-amplifying RNA.

Graphical abstract

Figure 1

Summary of in vitro transcribed repRNA constructs encoding HIV-1 Env genes and confirmation of protein expression by western blotting (a) Schematic of the TC-83 replicon structure showing the subgenome encoding the gene of interest (GOI). HIV-1 genes were cloned in the TC-83 subgenome, and repRNA constructs were synthesized by in vitro transcribed reactions. (b) In vitro transcribed RNA constructs were analyzed by agarose gel electrophoresis to verify molecular weight and molecule integrity. (c–e) Western blot analyses of cell lysates and supernatants collected from BHK21 cells transfected with synthesized constructs. Gels were labeled with anti-gp120 human monoclonal antibody (mAb) 2G12, anti-gp41 human mAb 4E10 or anti-HIV-1 rabbit polyclonal against Gag polyprotein. pos., recombinant BG505 SOSIP.664 used as a positive control; neg., repRNA-encoding RFP as a negative control; A + B, co-transfection with constructs A and B. All samples were run on the same RNA (b) or protein (c–e) gel. Where applicable, black dividing lines are shown to exclude an irrelevant sample run in between constructs C and D.

Figure 2

Comparison of anti-gp140 IgG responses induced by various HIV-1 repRNA constructs in mouse and rabbit models Serum anti-gp140 IgG responses in (A) female C57Bl/6 mice (6–8 weeks old; n = 5/group) and (B) young adult female New Zealand White (NZW) rabbits (approximately 28 weeks old; n = 4/group) after receiving two doses 3 weeks apart. (A) Mice in each group were immunized by intramuscular injection with 1 μg of LION-formulated repRNA construct indicated on the vertical axis. Mice immunized with repRNA expressing Env FL, BG505 SOSIP.664-scfl, or Gag-IRES-Env were partially seropositive and advanced to rabbit immunogenicity study shown in (B). NZW rabbits were immunized by intramuscular injection with 100 μg or 50 μg of each candidate repRNA formulated with LION. Serum IgG responses in mice and rabbits against the BG505 SOSIP.664 gp140 recombinant protein were measured by ELISA. Individual and mean (SD) endpoint titers are shown. Mean endpoint titer from each group in (A) was log₁₀ transformed and compared against the Gag group by ordinary one-way ANOVA and Dunnett’s multiple comparisons test. Mean endpoint titer from each group in (B) was log₁₀ transformed and compared against every other group by ordinary one-way ANOVA and Tukey’s multiple comparisons test. Statistically significant differences in (A) are only shown for groups with at least one seropositive animal and in (B) only shown for each dose cohort. Summary of p values: ∗ < 0.05; ∗∗∗ < 0.001; ∗∗∗∗ < 0.0001.

Figure 3

Overview of timeline and study design conducted in pregnant rabbits and newborn kits (A) Study timeline to evaluate maternal vaccination in pregnant NZW female rabbits and newborn kits. (B) Matrixed study design showing allocation of kits from each litter to vaccination group. Numbers in parentheses indicate sample size.

Figure 4

Mean (±SD) (A) weight change in pregnant rabbits and (B) corresponding litter sizes as a measure of tolerogenicity. There were no significant differences in litter sizes between groups compared by ordinary one-way ANOVA and Dunnett's multiple comparisons test.

Figure 5

Vaccine-induced serum IgG kinetics in NZW female adult rabbits Rabbits (n = 5 per group) received (A) saline, (B) RNA-prM/E, (C) RNA-SOSIP-scfl-scfl, or (D) Protein-SOSIP. Animals in both RNA groups (B and C) received 100 μg of repRNA formulated with LION and in the protein group (D) received 50 μg of recombinant BG505-SOSIP.664 trimer adjuvanted with the squalene emulsion adjuvant AddaVax. Sera were assayed by antigen-specific ELISA to measure binding antibodies against (top panels) BG505 SOSIP.664 gp140 or (bottom panels) ZIKV E protein. Plotted lines track the binding antibody response of a single animal corresponding to the unique tag number in the legend. Gray shaded regions mark periods when rabbits were mated (week 1) and when kits were born (week 6–7). Arrows indicate immunization time points.

Figure 6

RNA-prM/E induced antigen-specific binding and neutralizing antibody responses in pregnant rabbits Sera collected on weeks 7 and 13 after primary immunization were assayed for (A and B) anti-ZIKV E IgG responses by ELISA and (C) ZIKV (PRVABC56 isolate) neutralization by measuring cytopathic effect (CPE) in Vero E6 cells using the xCELLigence system. (D) Correlation plot between binding and neutralizing responses showing Pearson’s correlation coefficient (r value) and significance of correlation (p value). Mean endpoint titer between RNA-prM/E and RNA-SOSIP-scfl groups was compared using non-parametric Mann-Whitney test (∗∗p < 0.005).

Figure 7

Summary of serum HIV-1 antibody responses in pregnant NZW rabbits on week 7 (3 weeks post second immunization) and week 13 (2 weeks post third immunization) (A) Individual and mean endpoint binding IgG titers against BG505 SOSIP.664 gp140 were measured by ELISA. (B) Individual and mean neutralizing antibodies shown as percent neutralization of autologous BG505.T332N pseudovirus in TZM-bl cells. RNA-SOSIP-scfl and Protein-SOSIP groups were compared using Mann-Whitney test (∗p < 0.05; ns, not significant).

Figure 8

Newborn kits acquired antibodies in utero from vaccinated does Individual and mean (±SD) (A) anti-gp140 and (B) anti-ZIKV E IgG titers in kits near birth showed that passively acquired antibodies are antigen specific and depend on the vaccine status of does. In (A), log₁₀ transformed mean endpoint titers of RNA-SOSIP-scfl and Protein-SOSIP groups were compared by Mann-Whitney test (∗two-tailed p value <0.05). Mean endpoint titer of each group in (B) not immunized with a ZIKV E antigen was compared with the mean IgG titer in the RNA-prM/E group by ordinary one-way ANOVA and Dunnett’s multiple comparisons test (∗∗∗∗p < 0.0001). Vaccine-induced (C) anti-gp140 and (D) anti-ZIKV E antibodies showed strong positive correlation between week-7 IgG levels in does and corresponding kits at time of birth.

Figure 9

Newborns from RNA-prM/E vaccinated does acquired ZIKV neutralizing antibodies in utero that were directly proportional to neutralizing antibody levels in does (A) Sera from newborns collected at time of delivery between weeks 6 and 7 were assayed for ZIKV (PRVABC56 isolate) neutralization by measuring CPE in Vero E6 cells using the xCELLigence system. Mean serum dilution resulting in 50% neutralization between indicated groups was log₁₀ transformed and compared by ordinary one-way ANOVA and Tukey’s multiple comparisons test (∗∗∗∗p < 0.0001). (B) Correlation plot showing Pearson’s correlation coefficient (r value) and significance of correlation (p value) between neutralizing responses in does and kits.

Figure 10

Vaccination in kits induced higher mean antibody titers compared with unvaccinated kits regardless of doe vaccination status Antibody responses shown here are from 4 weeks after booster immunization. Each cohort of newborns delivered by does in the same group (vertical axis labels) was randomized and received RNA-prM/E, RNA-SOSIP-scfl, Protein-SOSIP, or saline (boxed legend). Mean (±SD) and individual endpoint binding IgG titers in kits measured by ELISA against (A) gp140 and (B) ZIKV E antigens. Statistical analyses performed on log₁₀ transformed data. Means of vaccinated and unvaccinated (saline) groups were compared by two-way ANOVA and Dunnett’s multiple comparisons test. Only statistical significance is shown (∗p < 0.05, ∗∗p < 0.005, ∗∗∗∗p < 0.0001).

Figure 11

Comparison of vaccine-induced responses in the context of pre-existing maternal antibodies Serum anti-gp140 IgG endpoint titer in kits 4 weeks after receiving a booster immunization of (A) RNA-prM/E, (B) RNA-SOSIP-scfl or (C) Protein-SOSIP vaccine. Kits birthed from does receiving saline or RNA-prM/E are grouped together as negative (−) for maternal antibodies, and those birthed from does receiving RNA-SOSIP-scfl or Protein-SOSIP are grouped together as positive (+) for pre-existing maternal antibodies. Data were log₁₀ transformed and compared by Mann-Whitney test to measure statistical differences, which were not statistically significant (ns, p > 0.05).

Figure 12

Vaccination slowed passive waning of maternal antibodies Mean (±SD) IgG kinetics in kits with maternal antibodies against (A) ZIKV E or (B and C) gp140. Lines show fits by least-squares regression analysis. Mean (±SD) pre-existing antibody levels measured in euthanized newborn kits are shown to indicate the pre-vaccination antibody levels in each cohort of kits. Data arrows indicate weeks when kits in each cohort received RNA-prM/E, RNA-SOSIP-scfl, Protein-SOSIP, or saline (labels in legend). Vertical shaded regions indicate time frame when kits were weaned.

Figure 13

Physicochemical characteristics of LION formulation and LION/repRNA complexed vaccine Mean (±SD) (A) z-average particle size and (B) zeta potential characteristics before and after mixing LION and repRNA to form LION/repRNA complex at N:P ratio of 15. (C) Denaturing agarose gel electrophoresis was performed to assess binding of repRNA with LION, shown in lane labeled “LION™/repRNA complex (no PC),” where PC denotes phenol-chloroform extraction. As observed, no free repRNA was detected, and bound repRNA migrated toward the negatively charged electrode due to the net positive charge of the LION/repRNA complex. Also shown is the role of LION in repRNA protection against RNase-catalyzed hydrolysis. Naked (unformulated) repRNA or LION/repRNA complex was incubated in nuclease-free water (−) or RNases (+), followed by proteinase K inhibition and extraction with phenol-chloroform (PC). Compared with naked repRNA, LION-formulated repRNA was protected from RNases. All samples were run on the same gel.