Establishing RTS,S/AS01 as a benchmark for comparison to next-generation malaria vaccines in a mouse model.

New strategies are needed to reduce the incidence of malaria, and promising approaches include vaccines targeting the circumsporozoite protein (CSP). To improve upon the malaria vaccine, RTS,S/AS01, it is essential to standardize preclinical assays to measure the potency of next-generation vaccines against this benchmark. We focus on RTS,S/AS01-induced antibody responses and functional activity in conjunction with robust statistical analyses. Transgenic Plasmodium berghei sporozoites containing full-length P. falciparum CSP (tgPb-PfCSP) allow two assessments of efficacy: quantitative reduction in liver infection following intravenous challenge, and sterile protection from mosquito bite challenge. Two or three doses of RTS,S/AS01 were given intramuscularly at 3-week intervals, with challenge 2-weeks after the last vaccination. Minimal inter- and intra-assay variability indicates the reproducibility of the methods. Importantly, the range of this model is suitable for screening more potent vaccines. Levels of induced anti-CSP antibody 2A10 equivalency were also associated with activity: 105 µg/mL (95% CI: 68.8, 141) reduced liver infection by 50%, whereas 285 µg/mL (95% CI: 166, 404) is required for 50% sterile protection from mosquito bite challenge. Additionally, the liver burden model was able to differentiate between protected and non-protected human plasma samples from a controlled human malaria infection study, supporting these models' relevance and predictive capability. Comparison in animal models of CSP-based vaccine candidates to RTS,S/AS01 is now possible under well controlled conditions. Assessment of the quality of induced antibodies, likely a determinant of durability of protection in humans, should be possible using these methods.

Engineered DNA-encoded monoclonal antibodies targeting Plasmodium falciparum circumsporozoite protein confer single dose protection in a murine malaria challenge model.

Novel approaches for malaria prophylaxis remain important. Synthetic DNA-encoded monoclonal antibodies (DMAbs) are a promising approach to generate rapid, direct in vivo host-generated mAbs with potential benefits in production simplicity and distribution coupled with genetic engineering. Here, we explore this approach in a malaria challenge model. We engineered germline-reverted DMAbs based on human mAb clones CIS43, 317, and L9 which target a junctional epitope, major repeat, and minor repeat of the Plasmodium falciparum circumsporozoite protein (CSP) respectively. DMAb variants were encoded into a plasmid vector backbone and their expression and binding profiles were characterized. We demonstrate long-term serological expression of DMAb constructs resulting in in vivo efficacy of CIS43 GL and 317 GL in a rigorous mosquito bite mouse challenge model. Additionally, we engineered an Fc modified variant of CIS43 and L9-based DMAbs to ablate binding to C1q to test the impact of complement-dependent Fc function on challenge outcomes. Complement knockout variant DMAbs demonstrated similar protection to that of WT Fc DMAbs supporting the notion that direct binding to the parasite is sufficient for the protection observed. Further investigation of DMAbs for malaria prophylaxis appears of importance.

Evaluation of serological diagnostic tests of human brucellosis for prevention and control in Mexico.

Brucellosis is a zoonosis mainly present in developing countries. The WHO reports 500,000 new cases every year. From 2012 to 2016, 13,677 cases were reported in Mexico, with 2.00 to 2.64 rate per 100,000 inhabitants. To analyze the diagnostic algorithm of brucellosis in Mexico, we compared the commercial laboratory tests ELISA, Brucellacapt®, and lateral flow test (LFT) in a study of 473 individuals from two endemic Mexican populations. All patients were treated in first-level medical units for presenting brucellosis compatible symptoms and without a history of the disease. Clinical-epidemiological information was gathered and initial serum samples were obtained to react with anti-Brucella antibodies; subsequent samples were collected at follow-up treatment visits. Using the Rose Bengal screening, we found 165 negative samples and 308 positive reactive samples, of which 222 cases were confirmed and 234 were positive on at least one marker (IgG or IgM) or LFT. When Brucellacapt® was used, similar results to those observed with the conventional algorithm were found as judged by the Cohen's kappa coefficient (κ) (0.813, 95% CI 0.7788-0.8472). Similar κ indices between conventional algorithm and ELISA pair were found, 0.7038 (95% CI 0.6555-0.7521), representing high similarity between both groups of diagnosis. We conclude that conventional serodiagnoses, Brucellacapt® and LFT, presented inconclusive results and poor correlation between them. By contrast, ELISA test pair (IgG + IgM) presented high correlation with the conventional algorithm and greater capacity for correct positive and negative classification.

TcG2/TcG4 DNA Vaccine Induces Th1 Immunity Against Acute Trypanosoma cruzi Infection: Adjuvant and Antigenic Effects of Heterologous T. rangeli Booster Immunization.

Background: Chagas cardiomyopathy is caused by Trypanosoma cruzi (Tc). Two antigenic candidates, TcG2 and TcG4, are recognized by antibodies in naturally infected dogs and humans; and these vaccine candidates provided protection from Tc infection in mice and dogs. Trypanosoma rangeli (Tr) is non-pathogenic to mammals and shown to elicit cross-reactive anti-Tc antibodies. In this study, we investigated if fixed Tr (fTr) can further enhance the efficacy of the TcG2/TcG4 DNA vaccine. Methods and Results: C57BL/6 mice were immunized with TcG2/TcG4 DNA vaccine and fTr (delivered as an adjuvant or in prime-boost approach), and challenged with Tc. Serology studies showed that fTr (±quil-A) elicited Tc- and Tr-reactive IgGs that otherwise were not stimulated by TcG2/TcG4 vaccine only, and quil-A had suppressive effects on fTr-induced IgGs. After challenge infection, TcG2/TcG4-vaccinated mice exhibited potent expansion of antigen- and Tc-specific IgGs that were not boosted by fTr±quil-A. Flow cytometry analysis showed that TcG2/TcG4-induced dendritic cells (DC) and macrophages (Mφ) responded to challenge infection by expression of markers of antigen uptake, processing, and presentation, and production of pro-inflammatory cytokines. TcG2/TcG4-induced CD4+T cells acquired Th1 phenotype and expressed markers that orchestrate adaptive immunity. A fraction of vaccine-induced CD4+T cells exhibited iTreg phenotype responsible for aversion of self-injurious immune responses. Further, TcG2/TcG4-vaccinated mice exhibited potent expansion of poly-functional CD8+T cells with TNF-α/IFN-γ production and cytolytic phenotype post-infection. Subsequently, tissue parasites and pathology were hardly detectable in TcG2/TcG4-vaccinated/infected mice. Inclusion of fTr±quil-A had no clear additive effects in improving the Tc-specific adaptive immunity and parasite control than was noted in mice vaccinated with TcG2/TcG4 alone. Non-vaccinated mice lacked sufficient activation of Th1 CD4+/CD8+T cells, and exhibited >10-fold higher levels of tissue parasite burden than was noted in vaccinated/infected mice. Conclusion:TcG2/TcG4 vaccine elicits highly effective immunity, and inclusion of fTr is not required to improve the efficacy of DNA vaccine against acute Tc infection in mice.

A DNA vaccine encoding for TcSSP4 induces protection against acute and chronic infection in experimental Chagas disease.

Immunization of mice with plasmids containing genes of Trypanosoma cruzi induces protective immunity in the murine model of Chagas disease. A cDNA clone that codes for an amastigote-specific surface protein (TcSSP4) was used as a candidate to develop a DNA vaccine. Mice were immunized with the recombinant protein rTcSSP4 and with cDNA for TcSSP4, and challenged with bloodstream trypomastigotes. Immunization with rTcSSP4 protein makes mice more susceptible to trypomastigote infection, with high mortality rates, whereas mice immunized with a eukaryotic expression plasmid containing the TcSSP4 cDNA were able to control the acute phase of infection. Heart tissue of gene-vaccinated animals did not show myocarditis and tissue damage at 365 days following infection, as compared with control animals. INF-γ was detected in sera of DNA vaccinated mice shortly after immunization, suggesting the development of a Th1 response. The TcSSP4 gene is a promising candidate for the development of an anti-T. cruzi DNA vaccine.