Molecular adjuvant HMGB1 enhances anti-influenza immunity during DNA vaccination.

DNA-based vaccines, while highly immunogenic in mice, generate significantly weaker responses in primates. Therefore, current efforts are aimed at increasing their immunogenicity, which include optimizing the plasmid/gene, the vaccine formulation and method of delivery. For example, co-immunization with molecular adjuvants encoding an immunomodulatory protein has been shown to improve the antigen (Ag)-specific immune response. Thus, the incorporation of enhancing elements, such as these, may be particularly important in the influenza model in which high titered antibody (Ab) responses are critical for protection. In this regard, we compared the ability of plasmid-encoded high-mobility group box 1 protein (HMGB1), a novel cytokine in which we have previously mutated in order to increase DNA vaccine immunogenicity, with boost Ag-specific immune responses during DNA vaccination with influenza A/PR/8/34 nucleoprotein or the hemagglutinin of A novel H1N1/09. We show that the HMGB1 adjuvant is capable of enhancing adaptive effector and memory immune responses. Although Ag-specific antibodies were detected in all vaccinated animals, a greater neutralizing Ab response was associated with the HMGB1 adjuvant. Furthermore, these responses improved CD8 T+-cell effector and memory responses and provided protection against a lethal mucosal influenza A/PR/8/34 challenge. Thus, co-immunization with HMGB1 has strong in vivo adjuvant activity during the development of immunity against plasmid-encoded Ag.

Plasmids encoding the mucosal chemokines CCL27 and CCL28 are effective adjuvants in eliciting antigen-specific immunity in vivo.

A hurdle facing DNA vaccine development is the ability to generate strong immune responses systemically and at local immune sites. We report a novel systemically administered DNA vaccination strategy using intramuscular codelivery of CCL27 or CCL28, which elicited elevated peripheral IFN-gamma and antigen-specific IgG while driving antigen-specific T-cell secretion of cytokine and antibody production in the gut-associated lymphoid tissue and lung. This strategy resulted in induction of long-lived antibody responses that neutralized influenza A/PR8/34 and protected mice from morbidity and mortality associated with a lethal intranasal viral challenge. This is the first example of the use of CCL27 and CCL28 chemokines as adjuvants to influence a DNA vaccine strategy, suggesting further examination of this approach for manipulation of vaccine-induced immunity impacting both quality and phenotype of responses.

CCR10 expression is required for the adjuvant activity of the mucosal chemokine CCL28 when delivered in the context of an HIV-1 Env DNA vaccine.

An effective prophylactic vaccine targeting HIV must induce a robust humoral response and must direct the bulk of this response to the mucosa-the primary site of HIV transmission. The chemokine, CCL28, is secreted by epithelial cells at mucosal surfaces and recruits' cells expressing its receptor CCR10. CCR10 is predominantly expressed by IgA + ASCs. We hypothesized that co-immunization with plasmid DNA encoding consensus envelope antigens with plasmid-encoded CCL28 would enhance anti-HIV IgA responses at mucosal surfaces. Indeed, animals receiving pCCL28 and pEnvA/C had significantly increased HIV-specific IgA in fecal extract. Surprisingly, CCL28 co-immunization induced a significant increase in anti-HIV IgG in the serum in mice compared to those receiving pEnvA/C alone. These robust antibody responses were not associated with changes in the frequency of germinal center B cells but depended upon the expression of CCR10, as these responses we abolished in CCR10-deficient animals. Finally, immunization with CCL28 led to increased frequencies in HIV-specific CCR10 + and CCR10 + IgA + B cells in the small intestine and Peyer's patches of vaccinated animals as compared to those receiving pEnvA/C alone. These data indicate that CCL28 administration can enhance antigen-specific humoral responses systemically and at mucosal surfaces.

Human immunodeficiency virus type 1 (HIV-1) Vpr-regulated cell death: insights into mechanism.

The destruction of CD4(+) T cells and eventual induction of immunodeficiency is a hallmark of the human immunodeficiency virus type 1 infection (HIV-1). However, the mechanism of this destruction remains unresolved. Several auxiliary proteins have been proposed to play a role in this aspect of HIV pathogenesis including a 14 kDa protein named viral protein R (Vpr). Vpr has been implicated in the regulation of various cellular functions including apoptosis, cell cycle arrest, differentiation, and immune suppression. However, the mechanism(s) involved in Vpr-mediated apoptosis remains unresolved, and several proposed mechanisms for these effects are under investigation. In this review, we discuss the possibility that some of these proposed pathways might converge to modulate Vpr's behavior. Further, we also discuss caveats and future directions for investigation of the interesting biology of this HIV accessory gene.

HIV-1 Vpr regulates expression of beta chemokines in human primary lymphocytes and macrophages.

The HIV-1 vpr gene encodes a 14-kDa virion-packaged protein that has been implicated in viral pathogenesis. Vpr exhibits profound effects on human primary cells influencing proliferation, differentiation, apoptosis, and cytokine production, in part through NF-kappaB-mediated transcription. NF-kappaB, a potent transcription factor, activates many proinflammatory cytokines/chemokines upon infection. Here, we analyzed the effect of extracellular Vpr as well as the virion-associated Vpr on beta chemokines (MIP-1alpha, MIP-1beta, and RANTES) production in human macrophages and primary lymphocytes (PBLs). Macrophages and PBLs exposed to HIV-1 vpr+ viruses or to recombinant Vpr protein produced significantly less beta chemokines compared with cells infected with HIV-1 vpr-viruses or irrelevant control protein (Gag)-exposed cells. These results suggest that a Vpr-mediated increase in virus replication could be in part through down-regulation of chemokine production.

Immunogenicity of a novel enhanced consensus DNA vaccine encoding the leptospiral protein LipL45.

Leptospirosis is a bacterial zoonotic disease caused by an infection with a spirochete belonging to the genus Leptospira. In animals, leptospirosis displays a wide range of pathologies, including fever, abortion, icterus, and uveitis. Conversely, infection in humans is associated with multi-organ injury, resulting in an increased rate of fatalities. Pathogenic leptospires are able to translocate through cell monolayers at a rate significantly greater than that of non-pathogenic leptospires. Thus, vaccine approaches have been focused on targeting bacterial motility, lipopolysaccharides (LPSs), lipoproteins, outer-membrane proteins (OMPs) and other potential virulence factors. Previous studies have indicated that leptospiral proteins elicit long-lasting immunological memory in infected humans. In the study reported here, the efficacy of a synthetic consensus DNA vaccine developed against the Leptospira membrane lipoprotein LipL45 was tested. After in vivo electroporation (EP) mediated intramuscular immunization with a synthetic LipL45 DNA vaccine (pLipL45) immunized mice developed a significant cellular response along with the development of anti-LipL45-specific antibodies. Specifically, the pLipL45 vaccine induced a significant Th1 type immune response, indicated by the higher production of IL-12 and IFN-γ cytokines. The results presented here are the first demonstration that a LipL45 based DNA immunogen has potential as a anti-Leptospira vaccine.

Immunogenicity of a novel DNA vaccine cassette expressing multiple human immunodeficiency virus (HIV-1) accessory genes.

OBJECTIVE: To develop an HIV-1 accessory gene immunogen using a DNA vaccine approach. METHODS: HIV-1 accessory genes vif, vpu and nef were modified to express under the control of a single promoter with cellular proteolytic cleavage sites between the coding sequences (VVN-P). Immune responses induced by these constructs were evaluated in mice. RESULTS: DNA vaccine construct (pVVN-P) expressing Vif, Vpu and Nef was processed and the fusion protein was cleaved appropriately. Vif, Vpu and Nef as a fusion protein with proteolytic cleavage sites (VVN-P) is able to induce a significant level of cellular immune responses. We also observed that accessory genes Vif, Vpu and Nef (VVN-P) induced an effective T helper 1 proliferative response measured by cytokine production. Furthermore, expression cassette pVVN-P was able to induce cytotoxic T lymphocyte (CTL) responses against diverse HIV-1 viruses in infected target cells. CONCLUSION: We conclude that cell-mediated immune responses induced by accessory gene constructs from clade B may have a broader recognition of divergent HIV-1 viruses and should be further examined for both prophylactic and therapeutic vaccination schemes against HIV-1.

Vpr-GFP virion particle identifies HIV-infected targets and preserves HIV-1Vpr function in macrophages and T-cells.

Human immunodeficiency virus type 1 (HIV-1) is known for its ability to infect immune cells, including T-cells and macrophages. The 96-amino acid Vpr, a virion-associated protein, is essential for viral replication in monocytes/macrophages and increases viral replication in primary and established T-cell lines. The Vpr protein regulates a number of host cellular events, including proliferation, differentiation, apoptosis, cytokine production, and NF-kappaB-mediated transcription. Most of these functions have been analyzed using either endogenous Vpr protein or cells transfected with a Vpr expression plasmid. We developed a lentiviral vector complemented with a Vpr expression plasmid that results in viral particles packaged with Vpr protein. To facilitate identification of the target cells infected with the particles containing Vpr, we fused green fluorescent protein (GFP) with the Vpr open reading frame and analyzed the biology of this novel particle. Vpr itself is expressed as a 14-kDa protein; however, in vitro translation of the pVpr-GFP plasmid resulted in the expression of 39-kDa fusion protein. The fusion molecule exhibited the same activity in arresting the cell cycle in G2 as does the wildtype Vpr molecule. Subcellular localization of Vpr and Vpr-GFP by immunofluoresence in human and murine cell lines indicated that Vpr by itself or with the reporter GFP showed a perinuclear staining pattern. Replication kinetics showed no significant difference between Vpr-GFP and native complemented pseudovirus replication in a single-round infectivity assay. A flow cytometry analysis of peripheral blood lymphocytes and macrophages infected with Vpr-GFP-packaged virions and selected by GFP showed 56.7% infectivity for lymphocytes and 84.6% infectivity for macrophages. Additional analysis of CD24 (HSA)-positive cells showed infection of CD4+ cells, macrophages, and, importantly, dendritic cells. This system will allow us to identify specific cell populations including antigen-presenting cells, and allow quantitative analysis of the precise effect of Vpr on both target and bystander cells in vitro as well as in vivo.

Novel and enhanced anti-melanoma DNA vaccine targeting the tyrosinase protein inhibits myeloid-derived suppressor cells and tumor growth in a syngeneic prophylactic and therapeutic murine model.

Melanoma is the most deadly type of skin cancer, constituting annually ∼ 75% of all cutaneous cancer-related deaths due to metastatic spread. Currently, because of metastatic spread, there are no effective treatment options for late-stage metastatic melanoma patients. Studies over the past two decades have provided insight into several complex molecular mechanisms as to how these malignancies evade immunological control, indicating the importance of immune escape or suppression for tumor survival. Thus, it is essential to develop innovative cancer strategies and address immune obstacles with the goal of generating more effective immunotherapies. One important area of study is to further elucidate the role and significance of myeloid-derived suppressor cells (MDSCs) in the maintenance of the tumor microenvironment. These cells possess a remarkable ability to suppress immune responses and, as such, facilitate tumor growth. Thus, MDSCs represent an important new target for preventing tumor progression and escape from immune control. In this study, we investigated the role of MDSCs in immune suppression of T cells in an antigen-specific B16 melanoma murine system utilizing a novel synthetic tyrosinase (Tyr) DNA vaccine therapy in both prophylactic and therapeutic models. This Tyr vaccine induced a robust and broad immune response, including directing CD8 T-cell infiltration into tumor sites. The vaccine also reduced the number of MDSCs in the tumor microenvironment through the downregulation of monocyte chemoattractant protein 1, interleukin-10, CXCL5 and arginase II, factors important for MDSC expansion. This novel synthetic DNA vaccine significantly reduced the melanoma tumor burden and increased survival in vivo, due likely, in part, to the facilitation of a change in the tumor microenvironment through MDSC suppression.

Molecular characterization of Chikungunya virus during an outbreak in South India.

INTRODUCTION: Re-emergence of Chikungunya is a major public health problem in the southern states of India. OBJECTIVES: This study was undertaken to investigate an outbreak of Chikungunya, in June-August 2008 using PCR and determine the prevalent genotypes of Chikungunya virus (CHIKV) associated with the outbreak. MATERIALS AND METHODS: Samples of blood were collected (in heparinized vacutainer tubes) from suspected patients of CHIKV infection from both Government Taluk Hospital in Kerala and a tertiary care hospital in Chennai, Tamil Nadu. A one-step RT-PCR was carried out on a block thermo-cycler targeting the E2 gene that codes for the viral envelope protein. The amplicons were verified for 305 bp size by standard agarose gel electrophoresis. The PCR products were purified, sequenced, and compared with other CHIKV strains reported from different geographical regions. A phylogenetic tree was constructed using MEGA 4. RESULTS: Altogether 118 samples were collected from patients who presented with sudden onset of fever and/or joint pain, myalgia, and headache. CHIKV infection was confirmed by RT-PCR in 14 patients and all these cases were from Kerala. The positivity correlated with the early stage of the disease as all these patients had fever of less than seven days duration. The study isolates have been allotted the GenBank accession nos. GQ272368-GQ272381. Phylogenetic analysis of recent CHIKV isolates by partial sequencing of E2 region shows that isolates are closely related to strains from neighboring states and the African type. CONCLUSION: RT-PCR is a useful technique for the early detection of CHIKV infection during outbreaks. Molecular characterization of the strains indicates that majority of the strains have originated from the Central/East African strains of CHIKV.

Juvenile hormone acid and ecdysteroid together induce competence for metamorphosis of the Verson's gland in Manduca sexta.

In the last larval instar of Lepidoptera, ecdysteroid in the absence of juvenile hormone (JH) is believed to cause the shift from larval to pupal development. In Manduca sexta, tissues such as the Verson's gland and crochet epidermis become pupally committed before the earliest pulse of ecdysteroid that occurs on day 2. What causes the change in commitment in these tissues? First it was necessary to determine at what stage these tissues become competent to express the pupal program. Last instar larvae of different ages were induced to molt prematurely by feeding the ecdysteroid analog RH5992 and Verson's gland proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Glands became competent to make pupal proteins between 24 and 32 h after the last larval ecdysis. Next, hormonal regulation of competence was examined in ligated abdomens of 12h last instar larvae. Treatment with JH II acid or methoprene acid plus a low dose (1/50th of the molt inducing dose) of RH5992 induced competence, whereas RH5992 alone, methoprene acid alone or methoprene plus RH5992 did not. Verson's glands maintained in vitro produced pupal proteins in response to methoprene acid together with RH5992 but not with RH5992 alone. Likewise, crochet epidermis lost the ability to make crochets (metamorphic change) only in isolated abdomens treated with JH II acid or methoprene acid and low doses of RH5992. In conclusion, JH acid in the presence of basal levels of ecdysteroid induces tissue competence for metamorphosis. Metamorphic competence is followed by commitment, induced by a small pulse of ecdysteroid in the absence of JH, and finally by expression caused by a high titer of ecdysteroid. It is proposed that JH acid is an essential metamorphic hormone.

Inclusion of Vpr accessory gene in a plasmid vaccine cocktail markedly reduces Nef vaccine effectiveness in vivo resulting in CD4 cell loss and increased viral loads in rhesus macaques.

We compared the immunogenicity of plasmid vaccines containing multiple human immunodeficiency virus (HIV) antigens and found that covaccination with plasmids expressing HIV-1 14 kDa vpr gene product profoundly reduces antigen-specific CD8-mediated cytotoxic T-cell activity (CTL). Interestingly, Th1 type responses against codelivered antigens (pGag-Pol, pNef, etc.) encoded by the plasmid vaccines were suppressed. This suggested that vpr might compromise CD8 T-cell immunity in vivo during infection. A pilot primate vaccine study was designed to test the hypothesis to compare the following groups: unvaccinated controls, animals vaccinated without simean immunodeficiency virus (SIV)-Nef antigen plasmid, and animals covaccinated with the identical plasmid antigen and a plasmid construct encoding SIV Vpr/Vpx. Animals were subsequently challenged intrarectally with pathogenic SIVmac251 after the final vaccination of a multiple immunization protocol. Control animals were all infected and exhibited high viral loads and rapid CD4+ T-cell loss. In contrast, the Nef plasmid-vaccinated animals were also infected but exhibited preservation of CD4+ T-cells and a multilog reduction in viral load compared with controls. Animals covaccinated multiple times with the Nef vaccine and pVpr/Vpx plasmid suffered rapid and profound loss of CD4+ T-cells. These results have important implications for the design of multicomponent and particle vaccines for HIV-1 as well as for our understanding of HIV/SIV pathogenesis in vivo.