Proteomic Basis of the Antibody Response to Monkeypox Virus Infection Examined in Cynomolgus Macaques and a Comparison to Human Smallpox Vaccination

Monkeypox is a zoonotic viral disease that occurs primarily in Central and West Africa. A recent outbreak in the United States heightened public health concerns for susceptible human populations. Vaccinating with vaccinia virus to prevent smallpox is also effective for monkeypox due to a high degree of sequence conservation. Yet, the identity of antigens within the monkeypox virus proteome contributing to immune responses has not been described in detail. We compared antibody responses to monkeypox virus infection and human smallpox vaccination by using a protein microarray covering 92–95% (166–192 proteins) of representative proteomes from monkeypox viral clades of Central and West Africa, including 92% coverage (250 proteins) of the vaccinia virus proteome as a reference orthopox vaccine. All viral gene clones were verified by sequencing and purified recombinant proteins were used to construct the microarray. Serum IgG of cynomolgus macaques that recovered from monkeypox recognized at least 23 separate proteins within the orthopox proteome, while only 14 of these proteins were recognized by IgG from vaccinated humans. There were 12 of 14 antigens detected by sera of human vaccinees that were also recognized by IgG from convalescent macaques. The greatest level of IgG binding for macaques occurred with the structural proteins F13L and A33R, and the membrane scaffold protein D13L. Significant IgM responses directed towards A44R, F13L and A33R of monkeypox virus were detected before onset of clinical symptoms in macaques. Thus, antibodies from vaccination recognized a small number of proteins shared with pathogenic virus strains, while recovery from infection also involved humoral responses to antigens uniquely recognized within the monkeypox virus proteome

Phosphotyrosine Substrate Sequence Motifs for Dual Specificity Phosphatases.

Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs) are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P) residue, but also the Ser(P) and Thr(P) residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7), atypical (DUSP3, DUSP14, DUSP22 and DUSP27), viral (variola VH1), and Cdc25 (A-C). Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P) peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets

Human Antibody Responses to Emerging Mayaro Virus and Cocirculating Alphavirus Infections Examined by Using Structural Proteins from Nine New and Old World Lineages

ABSTRACT Mayaro virus (MAYV), Venezuelan equine encephalitis virus (VEEV), and chikungunya virus (CHIKV) are vector-borne alphaviruses that cocirculate in South America. Human infections by these viruses are frequently underdiagnosed or misdiagnosed, especially in areas with high dengue virus endemicity. Disease may progress to debilitating arthralgia (MAYV, CHIKV), encephalitis (VEEV), and death. Few standardized serological assays exist for specific human alphavirus infection detection, and antigen cross-reactivity can be problematic. Therefore, serological platforms that aid in the specific detection of multiple alphavirus infections will greatly expand disease surveillance for these emerging infections. In this study, serum samples from South American patients with PCR- and/or isolation-confirmed infections caused by MAYV, VEEV, and CHIKV were examined by using a protein microarray assembled with recombinant capsid, envelope protein 1 (E1), and E2 from nine New and Old World alphaviruses. Notably, specific antibody recognition of E1 was observed only with MAYV infections, whereas E2 was specifically targeted by antibodies from all of the alphavirus infections investigated, with evidence of cross-reactivity to E2 of o’nyong-nyong virus only in CHIKV-infected patient serum samples. Our findings suggest that alphavirus structural protein microarrays can distinguish infections caused by MAYV, VEEV, and CHIKV and that this multiplexed serological platform could be useful for high-throughput disease surveillance. IMPORTANCE Mayaro, chikungunya, and Venezuelan equine encephalitis viruses are closely related alphaviruses that are spread by mosquitos, causing diseases that produce similar influenza-like symptoms or more severe illnesses. Moreover, alphavirus infection symptoms can be similar to those of dengue or Zika disease, leading to underreporting of cases and potential misdiagnoses. New methods that can be used to detect antibody responses to multiple alphaviruses within the same assay would greatly aid disease surveillance efforts. However, possible antibody cross-reactivity between viruses can reduce the quality of laboratory results. Our results demonstrate that antibody responses to multiple alphaviruses can be specifically quantified within the same assay by using selected recombinant protein antigens and further show that Mayaro virus infections result in unique responses to viral envelope proteins

Distribution of dephosphorylation data.

<p>(<b>A</b>) Representative results of Tyr(P) dephosphorylation of the peptide microarray library by VH1, the poxvirus DUSP. The scatter plot shows the relative florescence units (RFU) of the reference peptide microarray (red dots) ranked from high to low, and the RFUs for corresponding VH1-treated peptides (black dots). Each dot represents the average of triplicate values for one peptide in the library. (<b>B</b>) Untreated reference peptide microarray (red dots) compared with an overlay of quantile normalized results for each DUSP showing Tyr(P) peptide dephosphorylation (black dots). Peptides in each data set were sorted from high to low based on RFU.</p

Dual specificity phosphatases examined.

<p>^<i>a</i> Protein Data Base code</p><p>^<i>b</i> Hogan, M. et al., 2014 (submitted)</p><p>Dual specificity phosphatases examined.</p

Tyr(P) peptide microarray.

<p>(<b>A</b>) Coomassie blue staining of a SDS-PAGE gel showing the recombinant DUSP proteins examined. (<b>B</b>) Scanned images of DUSP treated Tyr(P) peptide microarrays. The human Tyr(P) peptides (>6000) were microarrayed in three identical subarrays on each slide. The microarrays were incubated with individual DUSPs and remaining Tyr(P) content was measured using anti- Tyr(P) monoclonal antibody and an Alexa-635 secondary (anti-mouse IgG) antibody. The control reference slide was treated with buffer only. The images were obtained from the same area of each slide. Each spot represents one peptide.</p

Structural comparison of DUSP3, DUSP14, DUSP22 and Cdc25B catalytic sites.

<p>(<b>A</b>) Superimposed ribbon representation of structures for DUSP3 (green), DUSP14 (magenta), DUSP22 (cyan) and Cdc25B (yellow). The catalytic sites are centered on the co-crystallized phosphate or 2-(N-morpholino) ethanesulfonic acid (MES) atom. (<b>B</b>) Electrostatic potential surface representation of the catalytic site of DUSP3 (PDB: 1VHR), DUSP14 (PDB: 2WGP), DUSP22 (PDB: 1WRM) and Cdc25B (PDB: 1QB0). Red and blue colored regions denote negative and positive charges, respectively.</p