Publications

Anaplasma phagocytophilum Major surface protein 4 (MSP4) plays a role during infection and multiplication in host neutrophils and tick vector cells. Recently, vaccination trials with the A. phagocytophilum antigen MSP4 in sheep showed only partial protection against pathogen infection. However, in rabbits immunized with MSP4, this recombinant antigen was protective. Differences between rabbit and sheep antibody responses are probably associated with the recognition of non-protective epitopes by IgG of immunized lambs. To address this question, we applied quantum vaccinomics to identify and characterize MSP4 protective epitopes by a microarray epitope mapping using sera from vaccinated rabbits and sheep. The identified candidate protective epitopes or immunological quantum were used for the design and production of a chimeric protective antigen. Inhibition assays of A. phagocytophilum infection in human HL60 and Ixodes scapularis tick ISE6 cells evidenced protection by IgG from sheep and rabbits immunized with the chimeric antigen. These results supported that the design of new chimeric candidate protective antigens using quantum vaccinomics to improve the protective capacity of antigens in multiple hosts.

Introduction The present work sought to identify MHC-I-restricted peptide signatures for arbovirus using in silico and in vitro peptide microarray tools. Methods First, an in-silico analysis of immunogenic epitopes restricted to four of the most prevalent human MHC class-I was performed by identification of MHC affinity score. For that, more than 10,000 peptide sequences from 5 Arbovirus and 8 different viral serotypes, namely Zika (ZIKV), Dengue (DENV serotypes 1-4), Chikungunya (CHIKV), Mayaro (MAYV) and Oropouche (OROV) viruses, in addition to YFV were analyzed. Haplotype HLA-A*02.01 was the dominant human MHC for all arboviruses. Over one thousand HLA-A2 immunogenic peptides were employed to build a comprehensive identity matrix. Intending to assess HLAA*02:01 reactivity of peptides in vitro, a peptide microarray was designed and generated using a dimeric protein containing HLA-A*02:01. Results The comprehensive identity matrix allowed the identification of only three overlapping peptides between two or more flavivirus sequences, suggesting poor overlapping of virus-specific immunogenic peptides amongst arborviruses. Global analysis of the fluorescence intensity for peptide-HLA-A*02:01 binding indicated a dose-dependent effect in the array. Considering all assessed arboviruses, the number of DENV-derived peptides with HLA-A*02:01 reactivity was the highest. Furthermore, a lower number of YFV-17DD overlapping peptides presented reactivity when compared to non-overlapping peptides. In addition, the assessment of HLA-A*02:01-reactive peptides across virus polyproteins highlighted non-structural proteins as “hot-spots”. Data analysis supported these findings showing the presence of major hydrophobic sites in the final segment of non-structural protein 1 throughout 2a (Ns2a) and in nonstructural proteins 2b (Ns2b), 4a (Ns4a) and 4b (Ns4b). Discussion To our knowledge, these results provide the most comprehensive and detailed snapshot of the immunodominant peptide signature for arbovirus with MHC-class I restriction, which may bring insight into the design of future virus-specific vaccines to arboviruses and for vaccination protocols in highly endemic areas.

Abstract Background Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe flu like symptoms. The acute symptoms disappear after one week, but chronic arthralgia can persist for years. Here, humoral immune responses in CHIKV-infected patients and vaccinees were analyzed. Methods Alphavirus neutralization activity was analyzed with pseudotyped lentiviral vectors and antibody epitope mapping was performed with a peptide array. Results Greatest CHIKV neutralization activity was observed 60-92 days after onset of symptoms. The amount of CHIKV-specific antibodies, their binding avidity and cross-reactivity with other alphaviruses increased over time. CHIKV and o’nyong-nyong virus (ONNV) were both neutralized to a similar extent. Linear antibody binding epitopes were mainly found in E2 domain B and the acid-sensitive regions (ASRs). In addition, serum samples from healthy volunteers vaccinated with a measles-vectored chikungunya vaccine candidate, MV-CHIK, were analyzed. Neutralization activity in the samples from the vaccine cohort was 2–6-fold lower than in samples from CHIKV-infected patients. In contrast to infection, vaccination only induced cross-neutralization with ONNV and the E2 ASR1 was the major antibody target. Conclusion These data could assist vaccine design and enable the identification of correlates of protection necessary for vaccine efficacy.

Background Vivax malaria is the predominant form of malaria outside Africa, affecting about 14 million people worldwide, with about 2.5 billion people exposed. Development of a Plasmodium vivax vaccine is a priority, and merozoite surface protein 7 (MSP-7) has been proposed as a plausible candidate. The P. vivax genome contains 12 MSP-7 genes, which contribute to erythrocyte invasion during blood-stage infection. Previous analysis of MSP-7 sequence diversity suggested that not all paralogs are functionally equivalent. To explore MSP-7 functional diversity, and to identify the best vaccine candidate within the family, MSP-7 expression and antigenicity during bloodstream infections were examined directly from clinical isolates. Methods Merozoite surface protein 7 gene expression was profiled using RNA-seq data from blood samples isolated from ten human patients with vivax malaria. Differential expression analysis and co-expression cluster analysis were used to relate PvMSP-7 expression to genetic markers of life cycle stage. Plasma from vivax malaria patients was also assayed using a custom peptide microarray to measure antibody responses against the coding regions of 12 MSP-7 paralogs. Results Ten patients presented diverse transcriptional profiles that comprised four patient groups. Two MSP-7 paralogs, 7A and 7F, were expressed abundantly in all patients, while other MSP-7 genes were uniformly rare (e.g. 7J). MSP-7H and 7I were significantly more abundant in patient group 4 only, (two patients having experienced longer patency), and were co-expressed with a schizont-stage marker, while negatively associated with liver-stage and gametocyte-stage markers. Screening infections with a PvMSP-7 peptide array identified 13 linear B-cell epitopes in five MSP-7 paralogs that were recognized by plasma from all patients. Conclusions These results show that MSP-7 family members vary in expression profile during blood infections; MSP-7A and 7F are expressed throughout the intraerythrocytic development cycle, while expression of other paralogs is focused on the schizont. This may reflect developmental regulation, and potentially functional differentiation, within the gene family. The frequency of B-cell epitopes among paralogs also varies, with MSP-7A and 7L consistently the most immunogenic. Thus, MSP-7 paralogs cannot be assumed to have equal potential as vaccines. This analysis of clinical infections indicates that the most abundant and immunogenic paralog is MSP-7A.

Matrix (M) protein of Newcastle disease virus (NDV) is an abundant protein that can induce a robust humoral immune response. However, its antigenic epitopes remain unknown. In this study, we used a pepscan approach to map linear B cell immunodominant epitopes (IDEs) of M protein with NDV-specific chicken antisera. The six epitopes with the highest reactivity by peptide scanning were obtained as IDE candidates. Among them, aa71–85 and aa349–363 were identified by immunological assays with NDV-specific or IDE-specific antisera. The minimal antigenic epitopes of the two IDEs were further characterized as 77MIDDKP82 and 354HTLAKYNPFK363. Moreover, an amino acid sequence alignment and immunoblot analysis revealed the conservation of the two IDEs in the M protein of strains of different genotypes. These two IDEs of M protein could be genetically eliminated as negative markers in recombinant NDV for serologically differential diagnosis in the development of marker vaccines.