Specific Biomarkers Associated With Neurological Complications and Congenital Central Nervous System Abnormalities From Zika Virus-Infected Patients in Brazil.

Background: Zika virus (ZIKV) infections have been linked to different levels of clinical outcomes, ranging from mild rash and fever to severe neurological complications and congenital malformations. Methods: We investigated the clinical and immunological response, focusing on the immune mediators profile in 95 acute ZIKV-infected adult patients from Campinas, Brazil. These patients included 6 pregnant women who later delivered during the course of this study. Clinical observations were recorded during hospitalization. Levels of 45 immune mediators were quantified using multiplex microbead-based immunoassays. Results: Whereas 11.6% of patients had neurological complications, 88.4% displayed mild disease of rash and fever. Several immune mediators were specifically higher in ZIKV-infected patients, and levels of interleukin 10, interferon gamma-induced protein 10 (IP-10), and hepatocyte growth factor differentiated between patients with or without neurological complications. Interestingly, higher levels of interleukin 22, monocyte chemoattractant protein 1, TNF-α, and IP-10 were observed in ZIKV-infected pregnant women carrying fetuses with fetal growth-associated malformations. Notably, infants with congenital central nervous system deformities had significantly higher levels of interleukin 18 and IP-10 but lower levels of hepatocyte growth factor than those without such abnormalities born to ZIKV-infected mothers. Conclusions: This study identified several key markers for the control of ZIKV pathogenesis. This will allow a better understanding of the molecular mechanisms of ZIKV infection in patients.

A promiscuous T cell epitope-based HIV vaccine providing redundant population coverage of the HLA class II elicits broad, polyfunctional T cell responses in nonhuman primates.

Over the last few decades, several emerging or reemerging viral diseases with no readily available vaccines have ravaged the world. A platform to fastly generate vaccines inducing potent and durable neutralizing antibody and T cell responses is sorely needed. Bioinformatically identified epitope-based vaccines can focus on immunodominant T cell epitopes and induce more potent immune responses than a whole antigen vaccine and may be deployed more rapidly and less costly than whole-gene vaccines. Increasing evidence has shown the importance of the CD4+ T cell response in protection against HIV and other viral infections. The previously described DNA vaccine HIVBr18 encodes 18 conserved, promiscuous epitopes binding to multiple HLA-DR-binding HIV epitopes amply recognized by HIV-1-infected patients. HIVBr18 elicited broad, polyfunctional, and durable CD4+and CD8+ T cell responses in BALB/c and mice transgenic to HLA class II alleles, showing cross-species promiscuity. To fully delineate the promiscuity of the HLA class II vaccine epitopes, we assessed their binding to 34 human class II (HLA-DR, DQ, and -DP) molecules, and immunized nonhuman primates. Results ascertained redundant 100% coverage of the human population for multiple peptides. We then immunized Rhesus macaques with HIVBr18 under in vivo electroporation. The immunization induced strong, predominantly polyfunctional CD4+ T cell responses in all animals to 13 out of the 18 epitopes; T cells from each animal recognized 7-11 epitopes. Our results provide a preliminary proof of concept that immunization with a vaccine encoding epitopes with high and redundant coverage of the human population can elicit potent T cell responses to multiple epitopes, across species and MHC barriers. This approach may facilitate the rapid deployment of immunogens eliciting cellular immunity against emerging infectious diseases, such as COVID-19.

Purified herpes simplex type 1 glycoprotein D (gD) genetically fused with the type 16 human papillomavirus E7 oncoprotein enhances antigen-specific CD8+ T cell responses and confers protective antitumor immunity.

Type 1 herpes virus (HSV-1) glycoprotein D (gD) enhances antigen-specific immune responses, particularly CD8(+) T cell responses, in mice immunized with DNA vaccines encoding hybrid proteins genetically fused with the target antigen at a site near the C-terminal end. These effects are attributed to the interaction of gD with the herpes virus entry mediator (HVEM) and the concomitant blockade of a coinhibitory mechanism mediated by the B- and T-lymphocyte attenuator (BTLA). However, questions concerning the requirement for endogenous synthesis of the antigen or the adjuvant/antigen fusion itself have not been addressed so far. In the present study, we investigated these points using purified recombinant gDs, genetically fused or not with type 16 papilloma virus (HPV-16) E7 oncoprotein. Soluble recombinant gDs, but not denatured forms, retained the ability to bind surface-exposed cellular receptors of HVEM-expressing U937 cells. In addition, in vivo administration of the recombinant proteins, particularly gD genetically fused with E7 (gDE7), promoted the activation of dendritic cells (DC) and antigen-specific cytotoxic CD8(+) T cells. More relevantly, mice immunized with the gDE7 protein developed complete preventive and partial therapeutic antitumor protection, as measured in mice following the implantation of TC-1 cells expressing HPV-16 oncoproteins. Collectively, these results demonstrate that the T cell adjuvant effects of the HSV-1 gD protein did not require endogenous synthesis and could be demonstrated in mice immunized with purified recombinant proteins.

The relationship between cytokine and neutrophil gene network distinguishes SARS-CoV-2-infected patients by sex and age.

The fact that the COVID-19 fatality rate varies by sex and age is poorly understood. Notably, the outcome of SARS-CoV-2 infections mostly depends on the control of cytokine storm and the increasingly recognized pathological role of uncontrolled neutrophil activation. Here, we used an integrative approach with publicly available RNA-Seq data sets of nasopharyngeal swabs and peripheral blood leukocytes from patients with SARS-CoV-2, according to sex and age. Female and young patients infected by SARS-CoV-2 exhibited a larger number of differentially expressed genes (DEGs) compared with male and elderly patients, indicating a stronger immune modulation. Among them, we found an association between upregulated cytokine/chemokine- and downregulated neutrophil-related DEGs. This was correlated with a closer relationship between female and young subjects, while the relationship between male and elderly patients was closer still. The association between these cytokine/chemokines and neutrophil DEGs is marked by a strongly correlated interferome network. Here, female patients exhibited reduced transcriptional levels of key proinflammatory/neutrophil-related genes, such as CXCL8 receptors (CXCR1 and CXCR2), IL-1ß, S100A9, ITGAM, and DBNL, compared with male patients. These genes are well known to be protective against inflammatory damage. Therefore, our work suggests specific immune-regulatory pathways associated with sex and age of patients infected with SARS-CoV-2 and provides a possible association between inverse modulation of cytokine/chemokine and neutrophil transcriptional signatures.

Anti-Flavivirus Vaccines: Review of the Present Situation and Perspectives of Subunit Vaccines Produced in Escherichia coli.

This article aims to review the present status of anti-flavivirus subunit vaccines, both those at the experimental stage and those already available for clinical use. Aspects regarding development of vaccines to Yellow Fever virus, (YFV), Dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) are highlighted, with particular emphasis on purified recombinant proteins generated in bacterial cells. Currently licensed anti-flavivirus vaccines are based on inactivated, attenuated, or virus-vector vaccines. However, technological advances in the generation of recombinant antigens with preserved structural and immunological determinants reveal new possibilities for the development of recombinant protein-based vaccine formulations for clinical testing. Furthermore, novel proposals for multi-epitope vaccines and the discovery of new adjuvants and delivery systems that enhance and/or modulate immune responses can pave the way for the development of successful subunit vaccines. Nonetheless, advances in this field require high investments that will probably not raise interest from private pharmaceutical companies and, therefore, will require support by international philanthropic organizations and governments of the countries more severely stricken by these viruses.

Febrile temperatures increase in vitro antibody affinity for malarial and dengue antigens.

Fever is a regulated increase of the body temperature resulting from both infectious and non-infectious causes. Fever is known to play a role in modulating immune responses to infection, but the potential of febrile temperatures in regulating antigen binding affinity to antibodies has not been explored. Here we investigated this process under in vitro conditions using Isothermal titration calorimetry and ELISA. We used selected malarial and dengue antigens against specific monoclonal antibodies, and observed a marked increase in the affinity of these antibody-antigen complexes at 40°C, compared to physiological (37°C) or pathophysiological temperatures (42°C). Induced thermal equilibration of the protein partners at these temperatures in vitro, prior to measurements, further increased their binding affinity. These results suggest another positive and adaptive role for fever in vivo, and highlight the favourable role of thermal priming in enhancing protein-protein affinity for samples with limited availability.

In vivo electroporation enhances vaccine-mediated therapeutic control of human papilloma virus-associated tumors by the activation of multifunctional and effector memory CD8+ T cells.

In vivo electroporation (EP) has reignited the clinical interest on DNA vaccines as immunotherapeutic approaches to control different types of cancer. EP has been associated with increased immune response potency, but its capacity in influencing immunomodulation remains unclear. Here we evaluated the impact of in vivo EP on the induction of cellular immune responses and therapeutic effects of a DNA vaccine targeting human papillomavirus-induced tumors. Our results demonstrate that association of EP with the conventional intramuscular administration route promoted a more efficient activation of multifunctional and effector memory CD8+ T cells with enhanced cytotoxic activity. Furthermore, EP increased tumor infiltration of CD8+ T cells and avoided tumor recurrences. Finally, our results demonstrated that EP promotes local migration of antigen presenting cells that enhances with vaccine co-delivery. Altogether the present evidences shed further light on the in vivo electroporation action and its impact on the immunogenicity of DNA vaccines.

Herpes Simplex Virus Glycoprotein D Targets a Specific Dendritic Cell Subset and Improves the Performance of Vaccines to Human Papillomavirus-Associated Tumors.

Cervical cancer is a major public health problem and one of the leading causes of cancer deaths in women. Virtually all cases of cervical cancer, as well as a growing share of anal and head/neck tumors, are associated with human papillomavirus (HPV) infection. Despite the effectiveness, the available prophylactic vaccines do not benefit women with cervical lesions or cancer. Therefore, the search of new immunotherapeutic approaches to treat HPV-induced tumors is still a priority. The present study characterizes a therapeutic antitumor vaccine based on the genetic fusion of the Herpes simplex virus-1 (HSV-1) glycoprotein D (gD) with the E7 oncoprotein from HPV-16 (gDE7). Two subcutaneous doses of gDE7, admixed with poly (I:C), conferred complete and long-lasting therapeutic antitumor protection on mice previously challenged with tumor cells expressing the HPV-16 oncoproteins. The vaccine induced multifunctional E7-specific CD8+ T cells with cytotoxic activity and effector memory phenotype (CD44+ CD62Llow). In addition, gDE7 admixed with poly (I:C) vaccination controlled the expansion of tumor-induced regulatory T cells and myeloid-derived suppressor cells. More importantly, gDE7 activated mouse CD11c+ CD8α+ and human BDCA3+ dendritic cells (DC), specialized in antigen cross-presentation to CD8+ T cells, under in vitro conditions. These results indicated that the activation of a specific DC population, mediated by gD, improved the antigen-specific immune responses and the therapeutic performance induced by antitumor vaccines. These results open perspectives for the clinical testing of gDE7-based vaccines under the concept of active immunization as a tool for the therapeutic control of cancer. Mol Cancer Ther; 16(9); 1922-33. ©2017 AACR.

Protection against HPV-16-Associated Tumors Requires the Activation of CD8+ Effector Memory T Cells and the Control of Myeloid-Derived Suppressor Cells.

Active anticancer immunotherapeutic approaches have been shown to induce cellular or humoral immune responses in patients, but, thus far, the observed outcomes did not ensure their recommendation for clinical use. The induction of tumor-specific CD8(+) T cells, although required for the clearance of most solid tumors, was shown to be insufficient for the development of a successful immunotherapeutic approach. The suppressive immune environment triggered by tumors, including the expansion of myeloid-derived suppressor cells (MDSC), is detrimental to the development of antitumor immune responses and precludes the generation of more promising clinical outcomes. In this work, we characterized the CD8(+) T-cell population specifically involved in the control of tumor growth and the role of MDSCs after administration of an antitumor therapeutic DNA vaccine targeting human papillomavirus type 16 (HPV-16)-associated tumors. Activation of cytotoxic high-avidity CD8(+) T cells with an effector memory phenotype was found in mice grafted with tumor cells expressing the HPV-16 oncoproteins. In addition, MDSC antibody depletion further enhanced the immunotherapeutic effects of the vaccine, resulting in the complete eradication of tumor cells. Collectively, the current results indicate that the simultaneous control of MDSCs and activation of high-avidity tumor-specific effector memory CD8(+) T cells are key features for tumor protection by immunotherapeutic approaches and deserve further testing under clinical conditions. Mol Cancer Ther; 15(8); 1920-30. ©2016 AACR.

Anti-flavivirus vaccines: review of the present situation and perspectives of subunit vaccines produced in escherichia coli

This article aims to review the present status of anti-flavivirus subunit vaccines, both those at the experimental stage and those already available for clinical use. Aspects regarding development of vaccines to Yellow Fever virus, (YFV), Dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) are highlighted, with particular emphasis on purified recombinant proteins generated in bacterial cells. Currently licensed anti-flavivirus vaccines are based on inactivated, attenuated, or virus-vector vaccines. However, technological advances in the generation of recombinant antigens with preserved structural and immunological determinants reveal new possibilities for the development of recombinant protein-based vaccine formulations for clinical testing. Furthermore, novel proposals for multi-epitope vaccines and the discovery of new adjuvants and delivery systems that enhance and/or modulate immune responses can pave the way for the development of successful subunit vaccines. Nonetheless, advances in this field require high investments that will probably not raise interest from private pharmaceutical companies and, therefore, will require support by international philanthropic organizations and governments of the countries more severely stricken by these viruses