Multi high-throughput approach for highly selective identification of vaccine candidates: the Group A Streptococcus case

Abstract

We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.

Fig. 1.

Flowchart of antigen selection strategy for vaccine candidate discovery. The three experimental approaches used are schematically shown in panels A and B. In C, a Venn diagram is represented, summarizing the expected characteristics of the antigens that are positive to at least one of the approaches used.

Fig. 2.

GAS antigen surface exposure and in vivo immunogenicity. A, Unsupervised hierarchical clustering of human pharyngitis patients sera (n = 239, x axis), versus the 61 antigens considered (y axis). Antigen/sera interactions resulting in signals with high or low fluorescence intensity (FI) are visualized in yellow and blue respectively. Color scale of signal intensity is reported on bottom-right. Antigens and/or sera showing similar reactivity profiles are grouped in clusters. The red bar on the left of the dendrogram identifies 26 antigens that were positively recognized by at least 50% of tested sera with an FI equal to or higher than the arbitrary cut-off of 15,000. B, The graph reports the percentage of FACS positivity for the 61 selected antigens against a panel of 22 strains. The red dashed line defines the 30% positivity value, which was considered the cutoff to choose the 24 antigens (green bars) with highest priority. Asterisks indicate those antigens displaying surface expression levels corresponding to the mean value plus > 2 S.D. in at least 1 tested strain.

Fig. 3.

The three-approach strategy identifies six top priority antigens. The Venn diagram reports the antigen distribution obtained on the basis of antigen positivity with the three technologies. Antigens highlighted in black are those conferring protection in the animal models against infection with M1 and M23 GAS isolates (Table I).

Fig. 4.

Immunization with the protein Combo confers consistent protection against infection with multiple GAS serotypes. Panels A and B show the survival rates of Combo immunized mice infected with four strains of different serotypes intranasally and intraperitoneally respectively. Survival of mice immunized either with adjuvant only (negative control groups) or with each of the four homologous M proteins (positive control groups) are also reported. Data for each group result from a minimum of three independent experiments with a total number of at least 32 mice. Asterisks indicate statistically significant difference between groups immunized either with adjuvant or with Combo (** = p value < 0.01; *** = p value <0.001, Fisher's exact test). The diagram in C reports the rate of bacterial growth (multiplication factor, y axis) in the air pouches of individual mice immunized with the protein Combo (black squares), adjuvant only (black circles) or M1 positive control protein (black triangles) and then infected in the pouch with the M1–3348 strain. A multiplication factor was obtained for each mouse as the ratio between the CFU number 24 h post-infection and time 0. Mann-Whitney U test was used for statistical analysis (*** = p value <0.001).

Fig. 5.

Immunization with the GAS protein Combo induces functional antibodies. A, Combo immune sera inhibit SPy0167-dependent hemolysis of sheep erythrocytes. Sheep blood cells were incubated with 10 ng of recombinant SPy0167 in the presence of increasing dilutions of sera from mice immunized with the alum-formulated Combo vaccine. Negative control reactions were incubated either without any serum or in the presence of serum from mice immunized with adjuvant only. B, Mice immunized with SPy0167 are protected by treatment with the toxin. The diagram shows the survival of mice immunized either with adjuvant alone or with SPy0167 following intravenous injection of increasing doses of recombinant SPy0167 (4–8 mice/group). C, Combo immune sera inhibit SPy0416-mediated processing of IL-8. The diagram in the upper panel shows the percent of uncleaved IL-8 in the presence of 100 ng of recombinant SPy0416 and different dilutions of Combo immune sera, as determined by ELISA assay. The results obtained with negative control samples without immune serum or with serum of mice immunized with adjuvant only are also shown. A silver-stained polyacrylamide gel showing the two IL-8 forms generated by cleavage is shown in the lower panel. D, Whole blood bactericidal assay with immune sera from Combo- and adjuvant-immunized rabbits. The data shown derive from 16 independent reactions set up using blood from three different naive rabbits incubated with rabbit immune sera. Statistically significant differences (*** = p value <0.0001) were obtained by Mann-Whitney analysis. Data are represented by box-and-whiskers plot analysis, showing the median, the 10 and 90 percentiles and extreme values (black dots).