Proof of concept for a single-dose Group B Streptococcus vaccine based on capsular polysaccharide conjugated to Qβ virus-like particles

Abstract

A maternal vaccine to protect neonates against Group B Streptococcus invasive infection is an unmet medical need. Such a vaccine should ideally be offered during the third trimester of pregnancy and induce strong immune responses after a single dose to maximize the time for placental transfer of protective antibodies. A key target antigen is the capsular polysaccharide, an anti-phagocytic virulence factor that elicits protective antibodies when conjugated to carrier proteins. The most prevalent polysaccharide serotypes conjugated to tetanus or diphtheria toxoids have been tested in humans as monovalent and multivalent formulations, showing excellent safety profiles and immunogenicity. However, responses were suboptimal in unprimed individuals after a single shot, the ideal schedule for vaccination during the third trimester of pregnancy. In the present study, we obtained and optimized self-assembling virus-like particles conjugated to Group B Streptococcus capsular polysaccharides. The resulting glyco-nanoparticles elicited strong immune responses in mice already after one immunization, providing pre-clinical proof of concept for a single-dose vaccine.

Fig. 1. Persitence of anti-PSII IgG titers in animals vaccinated with two doses of PSII-CRM and one dose of PSII-Qβ conjugates.

Serum samples were collected from groups of 10 immunized mice at times after one dose of PSII-Qβ or after one (day 21) or two doses of PSII-CRM conjugates. The geometric mean titer (RLU/mL) is indicated by the bars, individual mice are indicated by the dots, and the 95% Confidence Interval is indicated by the whiskers. For GMT, non responder sera were assigned titers half of the LLOQ. The Wilcoxon signed-ranks test was used to compare titers of the same immunization group and the Mann–Whitney test to compare different immunization groups. **P < 0.01; ****P < 0.0001. OPKA titers from pooled serum samples or each immunization group are reported below the histograms.

Fig. 2. Antibody responses in mice receiving GBS PSIa-CRM or -Qβ conjugates.

PSIa IgG titers in serum samples collected from mice (20 per group) receiving two doses of PSIa-CRM or one dose of PSIa-Qβ. The geometric mean titer (RLU/mL) is indicated by the bars, individual mice are indicated by the dots, and the 95% confidence interval is indicated by the whiskers. IgG titers between the two groups were compared by Mann–Whitney test. OPKA titers from pooled serum samples samples of each immunization group are reported below the barchart.

Fig. 3. Genetic strategy used for the generation of Qβ and Qβhp VLPs.

Top panel: expression of Qβ VLPs was achieved by cloning the Qβ coat protein open-reading frame under the T7 promoter; monomers self-assemble drandomly incorporating E.coli RNA. Bottom panel: for Qβhp VLPs expression, a hairpin structure was inserted immediately downstream of the stop codon of the gene encoding the Qβ coat protein; monomers self-assembled incorporating mainly Qβhp RNA.

Fig. 4. Sequencing analysis of RNA extracted from Qβ and Qβhp.

Quantification of the different categories of RNA-seq reads is indicated as a percentage of the total number of reads in samples from 1 lot of Qβ (first lane) and 2 lots of Qβhp (second and third lanes). In the bar plot, shades of blue are used for reads mapping to the E. coli BL21DE3 genomic sequence and shades of yellow and brown for reads mapping to the pET24b+ plasmid. lacI is reported in green as it is present in two copies on the genome and in one copy on the plasmid.

Fig. 5. Analytical characterization of PSII-Qβ (left) and PSII-Qβhp conjugates (right).

a Transmission electron microscopy (TEM) in negative staining, and b Size-exclusion High Performance Liquid Chromatography (SE-HPLC).

Fig. 6. Immune responses in mice receiving PSII conjugated to different Qβ VLPs.

Mice (10 per group) received one (full bars) or two doses (patterned bar) of PSII-CRM, PSII-Qβ, PSII-Qβhp, or RNAse-treated PSII-Qβhp. The geometric mean IgG titer (RLU/mL) is indicated by the bars, individual mice are indicated by the dots, and the 95% Confidence Interval is indicated by the whiskers. For GMT, non responder sera were assigned titers half of the LLOQ. The Wilcoxon signed-ranks test was used to compare titers at day 21 and day 42 of the same immunization group of the same immunization group and the Kruskal–Wallis and Dunn multiple comparisons test was used to compare different immunization groups. **P < 0.01. The corresponding OPKA titers from pools of serum samples are reported below the barchart.

Fig. 7. Immune responses in mice receiving PSII-CRM with different adjuvants.

Mice (10 per group) received a single dose of PSII-CRM formulated with AS04, Poly I:C, AS37 and CpG or PSII-Qβhp formulated with Aluminum hydroxide. The geometric mean IgG titer (RLU/mL) at day 21 and 42 is indicated by the bars, individual mice are indicated by the dots, and the 95% confidence interval is indicated by the whiskers. For GMT, non responder sera were assigned titers half of the LLOQ. The Kruskal–Wallis and Dunn multiple comparisons test was used to compare the group receiving PSII-Qβhp with all PSII-CRM immunization groups. **P < 0.01; ****P < 0.0001. The OPKA titers from pools of serum samples at day 21 are reported below the barchart.