Profiling the Targets of Protective CD8+ T Cell Responses to Infection.

T cells are critical effectors of host immunity that target intracellular pathogens, such as the causative agents of HIV, tuberculosis, and malaria. The development of vaccines that induce effective cell-mediated immunity against such pathogens has proved challenging; for tuberculosis and malaria, many of the antigens targeted by protective T cells are not known. Here, we report a novel approach for screening large numbers of antigens as potential targets of T cells. Malaria provides an excellent model to test this antigen discovery platform because T cells are critical mediators of protection following immunization with live sporozoite vaccines and the specific antigen targets are unknown. We generated an adenovirus array by cloning 312 highly expressed pre-erythrocytic Plasmodium yoelii antigens into adenovirus vectors using high-throughput methodologies. The array was screened to identify antigen-specific CD8+ T cells induced by a live sporozoite vaccine regimen known to provide high levels of sterile protection mediated by CD8+ T cells. We identified 69 antigens that were targeted by CD8+ T cells induced by this vaccine regimen. The antigen that recalled the highest frequency of CD8+ T cells, PY02605, induced protective responses in mice, demonstrating proof of principle for this approach in identifying antigens for vaccine development.

New gorilla adenovirus vaccine vectors induce potent immune responses and protection in a mouse malaria model.

BACKGROUND: A DNA-human Ad5 (HuAd5) prime-boost malaria vaccine has been shown to protect volunteers against a controlled human malaria infection. The potency of this vaccine, however, appeared to be affected by the presence of pre-existing immunity against the HuAd5 vector. Since HuAd5 seroprevalence is very high in malaria-endemic areas of the world, HuAd5 may not be the most appropriate malaria vaccine vector. This report describes the evaluation of the seroprevalence, immunogenicity and efficacy of three newly identified gorilla adenoviruses, GC44, GC45 and GC46, as potential malaria vaccine vectors. RESULTS: The seroprevalence of GC44, GC45 and GC46 is very low, and the three vectors are not efficiently neutralized by human sera from Kenya and Ghana, two countries where malaria is endemic. In mice, a single administration of GC44, GC45 and GC46 vectors expressing a murine malaria gene, Plasmodium yoelii circumsporozoite protein (PyCSP), induced robust PyCSP-specific T cell and antibody responses that were at least as high as a comparable HuAd5-PyCSP vector. Efficacy studies in a murine malaria model indicated that a prime-boost regimen with DNA-PyCSP and GC-PyCSP vectors can protect mice against a malaria challenge. Moreover, these studies indicated that a DNA-GC46-PyCSP vaccine regimen was significantly more efficacious than a DNA-HuAd5-PyCSP regimen. CONCLUSION: These data suggest that these gorilla-based adenovectors have key performance characteristics for an effective malaria vaccine. The superior performance of GC46 over HuAd5 highlights its potential for clinical development.

The CDI toxin of Yersinia kristensenii is a novel bacterial member of the RNase A superfamily.

Contact-dependent growth inhibition (CDI) is an important mechanism of inter-bacterial competition found in many Gram-negative pathogens. CDI+ cells express cell-surface CdiA proteins that bind neighboring bacteria and deliver C-terminal toxin domains (CdiA-CT) to inhibit target-cell growth. CDI+ bacteria also produce CdiI immunity proteins, which specifically neutralize cognate CdiA-CT toxins to prevent self-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiIYkris complex from Yersinia kristensenii ATCC 33638. CdiA-CTYkris adopts the same fold as angiogenin and other RNase A paralogs, but the toxin does not share sequence similarity with these nucleases and lacks the characteristic disulfide bonds of the superfamily. Consistent with the structural homology, CdiA-CTYkris has potent RNase activity in vitro and in vivo. Structure-guided mutagenesis reveals that His175, Arg186, Thr276 and Tyr278 contribute to CdiA-CTYkris activity, suggesting that these residues participate in substrate binding and/or catalysis. CdiIYkris binds directly over the putative active site and likely neutralizes toxicity by blocking access to RNA substrates. Significantly, CdiA-CTYkris is the first non-vertebrate protein found to possess the RNase A superfamily fold, and homologs of this toxin are associated with secretion systems in many Gram-negative and Gram-positive bacteria. These observations suggest that RNase A-like toxins are commonly deployed in inter-bacterial competition.

Identification of a suppressor mutation that improves the yields of hexon-modified adenovirus vectors.

We have generated hexon-modified adenovirus serotype 5 (Ad5) vectors that are not neutralized by Ad5-specific neutralizing antibodies in mice. These vectors are attractive for the advancement of vaccine products because of their potential for inducing robust antigen-specific immune responses in people with prior exposure to Ad5. However, hexon-modified Ad5 vectors displayed an approximate 10-fold growth defect in complementing cells, making potential vaccine costs unacceptably high. Replacing hypervariable regions (HVRs) 1, 2, 4, and 5 with the equivalent HVRs from Ad43 was sufficient to avoid Ad5 preexisting immunity and retain full vaccine potential. However, the resulting vector displayed the same growth defect as the hexon-modified vector carrying all 9 HVRs from Ad43. The growth defect is likely due to a defect in capsid assembly, since DNA replication and late protein accumulation were normal in these vectors. We determined that the hexon-modified vectors have a 32°C cold-sensitive phenotype and selected revertants that restored vector productivity. Genome sequencing identified a single base change resulting in a threonine-to-methionine amino acid substitution at the position equivalent to residue 342 of the wild-type protein. This mutation has a suppressor phenotype (SP), since cloning it into our Ad5 vector containing all nine hypervariable regions from Ad43, Ad5.H(43m-43), increased yields over the version without the SP mutation. This growth improvement was also shown for an Ad5-based hexon-modified vector that carried the hexon hypervariable regions of Ad48, indicating that the SP mutation may have broad applicability for improving the productivity of different hexon-modified vectors.