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. 2009 Dec 5;395(1):97-113.
doi: 10.1016/j.virol.2009.09.008. Epub 2009 Oct 2.

New classes of orthopoxvirus vaccine candidates by functionally screening a synthetic library for protective antigens

Alexandre Borovkov  1 , D Mitch Magee, Andrey Loskutov, Jose A Cano, Cheryl Selinsky, Jason Zsemlye, C Rick Lyons, Kathryn Sykes
Affiliations

New classes of orthopoxvirus vaccine candidates by functionally screening a synthetic library for protective antigens

Alexandre Borovkov et al. Virology. .

Abstract

The licensed smallpox vaccine, comprised of infectious vaccinia, is no longer popular as it is associated with a variety of adverse events. Safer vaccines have been explored such as further attenuated viruses and component designs. However, these alternatives typically provide compromised breadth and strength of protection. We conducted a genome-level screening of cowpox, the ancestral poxvirus, in the broadly immune-presenting C57BL/6 mouse as an approach to discovering novel components with protective capacities. Cowpox coding sequences were synthetically built and directly assayed by genetic immunization for open-reading frames that protect against lethal pulmonary infection. Membrane and non-membrane antigens were identified that partially protect C57BL/6 mice against cowpox and vaccinia challenges without adjuvant or regimen optimization, whereas the 4-pox vaccine did not. New vaccines might be developed from productive combinations of these new and existing antigens to confer potent, broadly efficacious protection and be contraindicated for none.

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Figures

Fig. 1
Fig. 1. Comparison of protection conferred by 4-pox gene vaccine in two strains of mice
Animals (10 mice per group) were biolistically immunized with a pool of VACV A27L, A33R, B5R, and L1R or the CPV homologues of these genes and boosted 2 weeks later. A lethal dose of virus (VACV strain Western Reserve, or CPV Brighten Red) was administered 6 weeks post prime by i.t. route, and survival was monitored for 21 days. Kaplan-Meir plots were drawn and relative to live virus vaccinated and naïve mice. A. The BALB/c mouse strain was used as host. B. The C57BL/6 mouse strain was used as host.
Fig. 2
Fig. 2. Evaluating immunogenicity of gene pools
A. In vivo activity of test-antigen expressing constructs. A mammalian expression vector (pCMViLS) carrying genes encoding antigens from cowpox (homologues of VACV A27L, A33R, B5R, and L1R), HSV-1 (gD), Y. pestis (V antigen) and anthrax (PA4) were biolistically co-delivered into the ears of a group of 10 C57BL/6 mice (2 × 1µg total DNA dose) at weeks 0 and 4. Two weeks after the boost, sera from the immunized group and a naïve control group were collected. Reactivities of pooled sera against each specific antigen were assayed by ELISA. Log serum dilution factors are plotted against fluorescence-units. For figure clarity, data on B5R, L1R, and V antigen were not plotted since they were not significantly different from naïve sera. B. Immune responses stimulated by the leader sequence fusion constructs within a complex pool of gene-expressing constructs. A pool of 250 ORFs from the genome of an irrelevant pathogen was spiked with the LS antigen expression constructs. A group of 10 mice were biolistically immunized (2 × 1µg shots) at weeks 0 and 4 with this complex pool and ORFs. Sera were drawn 2 weeks after the last boost, pooled and assayed against each of the four antigens by ELISA. Log serum dilution factors are plotted against fluorescence-unit readouts. C. Specific immune responses stimulated by ubiquitin fusion constructs within a complex pool of gene constructs. A pool of 250 ORFs from the genome of an irrelevant pathogen was spiked with the set of UB expression constructs. Mice (10 per group) were gene-gun immunized with the mixed inocula (2 × 1µg shots) at weeks 0 and 4. Pooled sera were analyzed and plotted as described in Figure 1B.
Fig. 3
Fig. 3. Individual sera analysis of pools of antigen-expressing test constructs
To discern responder frequencies, the sera from the mice described in Figure 2A and 2B were individually tested by fluorescent ELISA. Reactivities are shown against HSV1 glycoprotein (g)D in sera from mice immunized with the LS test constructs undiluted (A) or in mice immunized with the test constructs diluted into a pool of 250 irrelevant ORF expression constructs (B). Similarly, VACV A27L reactivity was measured in mice immunized with the LS test constructs undiluted (C) or diluted into the same pool of 250 irrelevant expression constructs (D).
Fig. 4
Fig. 4. ELI screen protection scores
Expression libraries built from these ORF pools were evaluated in a murine pulmonary challenge-protection assay. Each set of 25 groups represented the complete library and was designated X1–X25 (A), Y1–Y25 (B), or Z1–Z25 (C). C57BL/6 mice (10 per group) were challenged i.t. as described in Materials and Methods and monitored twice daily for 21 days. A protection score for each of the 75 groups was determined and plotted against controls. A27L/A33R/L1R/B5R is a mixed inoculum comprised of the cowpox homologues of the 4-component vaccinia vaccine candidate (4-pox). The eGFP plasmid expresses a codon-optimized green-fluorescence gene used as an irrelevant construct. Naïve and low-dose cowpox immunized groups are negative and positive controls, respectively. Groups significantly different from the negative control group are shown as gray bars. Note that for sets Y and Z, groups with significant protection are shown in gray. By contrast for set X, groups displaying enhanced disease are shown in gray. The 95% confidence interval relative to the irrelevant vaccine controls is shown. Nonparametric tests using the Mantel-Haenszel Logrank test showed identical or nearly so values. Risks significantly greater than the average odds-ratio were considered as candidates. All survival data were considered both parametrically and nonparametrically in order to prevent bias due to non-normal data distribution. Due to space consideration, survival plots are not shown.
Fig. 5
Fig. 5. Multiple ELI-selected ORFs map to the same genes
The genomic locations of the 36 individual CPV ORFs selected by matrix analysis of the protection assay results were determined. Sets of ORFs were identified that correspond to distinct but overlapping regions of the same gene. These genes are shown, represented as bold bars; the ORFs are drawn below the corresponding gene with its nucleotide endpoints indicated.
Fig. 6
Fig. 6. ORFs selected in the ELI screen were individually tested as vaccines in the challenge-protection assays
Analyses of the protection scores identified a shortlist of ORFs to further test as single-gene inocula. Groups of C57BL/6 mice (10 per group) were immunized with one plasmid, without adjuvant, and then i.t. challenged with a previously titrated lethal dose of CPV. Differences in cumulative survival hours were compared using the difference in means between control samples and test samples. Confidence intervals of the resulting difference in means (control minus test) that did not include 0 were considered significant and are colored grey. The dotted bar displays the upper limit of the 95% confidence interval for the test groups.
Fig. 7
Fig. 7. Confirmation of protective capacities of new C57BL/6 vaccine candidates
A. Survival of mice immunized with individual CPV gene vaccine candidates and challenged in an independently conducted protection experiment. Mantel- Haenszel survival plots (Prism, GraphPad Inc, LaJolla, CA) display differential survival rates of mice immunized with one of eight CPV vaccine candidates relative to mice immunized with a gene expressing an irrelevant vaccine antigen, F1-V. The calculated p values for each candidate are provided above the corresponding survival curve. Ten mice per group were used; an a posteriori power analysis indicates 6 mice per group were needed to observe differences in cumulative survival of less than 10% at 95% confidence and 80% power. B. Evaluation of a pool of the top nine ORF candidates in the vaccine assay. Mice (15 per group) were immunized with the ORFs identified in the ELI screen alongside positive and negative controls: live vaccine and naïve mice, respectively. Survival results following a lethal CPV challenge are displayed as Mantel- Haenszel plots. Data was analyzed as above.
Fig. 8
Fig. 8. T cell responses elicited by orthopox subunit vaccine candidates
A. Survey of peptide-stimulated T cell responses of mice individually immunized with the CPV vaccine candidates. Splenocytes from immunized mice were harvested and stimulated with a peptide pool comprised of 20-mers tiling the ORF-encoded product. IFNγ release of activated T cells was measured by ELISpot. Data are presented as the number of antigen-specific Spot Forming Units (SFU) per million splenocytes. SFU counts were adjusted for background by subtracting the number of spots in wells containing unstimulated splenocytes (no antigen). Triangular icons represent splenocyte results from individual mice. Black bars display group mean SFUs. Error bars reflect the standard deviation between quadruplicate assay wells. B. Comparison of peptide-stimulated T cell responses of mice immunized with a CPV ORF or a CPV homologue of 4-pox vaccine candidates. Splenocytes from immunized mice were harvested and stimulated as above. CPV homologues of the VACV 4-pox genes are as follow: CPV 165a/c (VACV A27L); CPV172 (VACV A33R); CPV 106 (VACV L1R); CPV 204 (VACV B5R). C. T cell cytokine release levels of candidate-immunized mice in response to protein-antigen stimulation. Splenocytes were stimulated with 20µg of recombinant protein, except for CPV100a-immune cells which were stimulated with peptide. IFNγ release of activated T cells was measured by ELISpot. Data are presented as the number of antigen-specific Spot Forming Units (SFU) per million splenocytes. SFU counts were adjusted for background by subtracting the number of spots in wells containing unstimulated splenocytes (no antigen). Icons represent splenocyte results from individual mice. Black bars display group mean SFUs. Error bars reflect the standard deviation between quadruplicate assay wells.
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