Karuppiah Muthumani

Chikungunya: A Potentially Emerging Epidemic?

Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005–07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts.

IMMUNOGENICITY OF NOVEL CONSENSUS-BASED DNA VACCINES AGAINST CHIKUNGUNYA VIRUS

Chikungunya virus (CHIKV) is an emerging arbovirus and is an important human pathogen. Infection of humans by CHIKV can cause a syndrome characterized by fever, headache, rash, nausea, vomiting, myalgia, arthralgia and occasionally neurological manifestations such as acute limb weakness. It is also associated with a fatal haemorrhagic condition. CHIKV is geographically distributed from Africa through Southeast Asia and South America, and its transmission to humans is mainly through the Aedes aegypti species mosquitoes. The frequency of recent epidemics in the Indian Ocean and La Reunion islands suggests that a new vector perhaps is carrying the virus, as A. aegypti are not found there. In fact, a relative the Asian tiger mosquito, Aedes albopictus, may be the culprit which has raised concerns in the world health community regarding the potential for a CHIK virus pandemic. Accordingly steps should be taken to develop methods for the control of CHIKV. Unfortunately, currently there is no specific treatment for Chikungunya virus and there is no vaccine currently available. Here we present data of a novel consensus-based approach to vaccine design for CHIKV, employing a DNA vaccine strategy. The vaccine cassette was designed based on CHIKV capsid- and envelope-specific consensus sequences with several modifications, including codon optimization, RNA optimization, the addition of a Kozak sequence, and a substituted immunoglobulin E leader sequence. The expression of capsid, envelope E1 and E1 was evaluated using T7-coupled transcription/translation and immunoblot analysis. A recently developed, adaptive constant-current electroporation technique was used to immunize C57BL/6 mice with an intramuscular injection of plasmid coding for the CHIK-Capsid, E1 and E2. Analysis of cellular immune responses, including epitope mapping, demonstrates that electroporation of these constructs induces both potent and broad cellular immunity. In addition, antibody ELISAs demonstrate that these synthetic immunogens are capable of inducing high titer antibodies capable of recognizing native antigen. Taken together, these data support further study of the use of consensus CHIK antigens in a potential vaccine cocktail.

HIV-1 Vpr Induces Apoptosis through Caspase 9 in T Cells and Peripheral Blood Mononuclear Cells

Human immunodeficiency virus, type 1 (HIV-1),vpr gene encodes a 14-kDa virion-associated protein, which exhibits significant effects on human cells. One important property of Vpr is its ability to induce apoptosis during infection. Apoptotic induction is likely to play a role in the pathogenesis of AIDS. However, the pathway of apoptosis is not clearly defined. In this report we investigate the mechanism of apoptosis induced by HIV-1 Vpr using a Vpr pseudotype viral infection system or adeno delivery of Vpr in primary human lymphoid cells and T-cells. With either vector, HIV-1 Vpr induced cell cycle arrest at the G2/M phase and apoptosis in lymphoid target cells. Furthermore, we observed that with both vectors, caspase 9, but not caspase 8, was activated following infection of human peripheral blood mononuclear cell with either Vpr-positive HIV virions or adeno-delivered Vpr. Activation of the caspase 9 pathway resulted in caspase 3 activation and apoptosis in human primary cells. These effects were coincident with the disruption of the mitochondrial transmembrane potential and induction of cytochrome c release by Vpr. The Vpr-induced signaling pathway did not induce CD95 or CD95L expression. Bcl-2 overexpressing cells succumb to Vpr-induced apoptosis. These studies illustrate that Vpr induces a mitochondria-dependent apoptotic pathway that is distinct from apoptosis driven by the Fas-FasL pathway.

Induction of Inflammation by West Nile virus Capsid through the Caspase-9 Apoptotic Pathway

West Nile virus (WNV) is a member of the Flaviviridae family of vector-borne pathogens. Clinical signs of WNV infection include neurologic symptoms, limb weakness, and encephalitis, which can result in paralysis or death. We report that the WNV-capsid (Cp) by itself induces rapid nuclear condensation and cell death in tissue culture. Apoptosis is induced through the mitochondrial pathway resulting in caspase-9 activation and downstream caspase-3 activation. Capsid gene delivery into the striatum of mouse brain or interskeletal muscle resulted in cell death and inflammation, likely through capsid-induced apoptosis in vivo. These studies demonstrate that the capsid protein of WNV may be responsible for aspects of viral pathogenesis through induction of the apoptotic cascade.

A DNA Vaccine against Chikungunya Virus Is Protective in Mice and Induces Neutralizing Antibodies in Mice and Nonhuman Primates

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines.

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

ObjectiveTo develop an HIV-1 accessory gene immunogen using a DNA vaccine approach. MethodsHIV-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. ResultsPDNA 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. ConclusionWe 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.

SIV DNA vaccine co-administered with IL-12 expression plasmid enhances CD8 SIV cellular immune responses in cynomolgus macaques

Abstract:  Current evidence suggests that a strong induced CD8 human immunodeficiency virus type 1 (HIV‐1)‐specific cell mediated immune response may be an important aspect of an HIV vaccine. The response rates and the magnitude of the CTL responses induced by current DNA vaccines in humans need to be improved and cellular immune responses to DNA vaccines can be enhanced in mice by co‐delivering DNA plasmids expressing immune modulators. Two reported to work well in the mouse systems are interleukin (IL)‐12 and CD40L. We sought to compare these molecular adjuvants in a primate model system. The cDNA for macaque IL‐12 and CD40L were cloned into DNA vectors. Groups of cynomolgus macaques were immunized with 2 mg of plasmid expressing SIVgag alone or in combination with either IL‐12 or CD40L. CD40L did not appear to enhance the cellular immune response to SIVgag antigen. However, more robust results were observed in animals co‐injected with the IL‐12 molecular adjuvant. The IL‐12 expanded antigen‐specific IFN‐γ positive effector cells as well as granzyme B production. The vaccine immune responses contained both a CD8 component as well a CD4 component. The adjuvanted DNA vaccines illustrate that IL‐12 enhances a CD8 vaccine immune response, however, different cellular profiles.

DNA vaccines for targeting bacterial infections

DNA vaccination has been of great interest since its discovery in the 1990s due to its ability to elicit both humoral and cellular immune responses. DNA vaccines consist of a DNA plasmid containing a transgene that encodes the sequence of a target protein from a pathogen under the control of a eukaryotic promoter. This revolutionary technology has proven to be effective in animal models and four DNA vaccine products have recently been approved for veterinary use. Although few DNA vaccines against bacterial infections have been tested, the results are encouraging. Because of their versatility, safety and simplicity a wider range of organisms can be targeted by these vaccines, which shows their potential advantages to public health. This article describes the mechanism of action of DNA vaccines and their potential use for targeting bacterial infections. In addition, it provides an updated summary of the methods used to enhance immunogenicity from codon optimization and adjuvants to delivery techniques including electroporation and use of nanoparticles.

Role of Proinflammatory Cytokines and Chemokines in Chronic Arthropathy in CHIKV Infection

Chikungunya virus (CHIKV) has caused large outbreaks worldwide in recent years. Acute-phase CHIKV infection has been reported to cause mild to severe febrile illness, and in some patients, this may be followed by long-lasting polyarthritis. The mainstay of treatment includes nonsteroidal anti-inflammatory drugs and other disease-modifying agents, the use of which is based on the assumption of an immunological interference mechanism in the pathogenesis. The present study has been designed to generate preliminary evidence to test this hypothesis. The levels of 30 cytokines were estimated in serum samples of acute CHIKV-infected patients, fully-recovered patients, patients with chronic CHIKV arthritis, and controls, using a quantitative multiplex bead ELISA. The levels of the proinflammatory cytokines IL-1 and IL-6 were elevated in acute patients, but IFN-γ/β and TNF-α levels remained stable. IL-10, which might have an anti-inflammatory effect, was also elevated, indicating a predominantly anti-inflammatory response in the acute phase of infection. Elevation of MCP-1, IL-6, IL-8, MIP-1α, and MIP-1β was most prominent in the chronic phase. These cytokines and chemokines have been shown to play important roles in other arthritides, including epidemic polyarthritis (EPA) caused by Ross River virus (RRV) and rheumatoid arthritis (RA).The immunopathogenesis of chronic CHIKV arthritis might have similarities to these arthritides. The novel intervention strategies being developed for EPA and RA, such as IL-6 and IL-8 signaling blockade, may also be considered for chronic CHIKV arthritis.

Mechanism of HIV-1 viral protein R-induced apoptosis ☆

The paradigm of HIV-1 infection includes the diminution of CD4(+) T cells, loss of immune function, and eventual progression to AIDS. However, the mechanisms that drive host T cell depletion remain elusive. One HIV protein thought to participate in this destructive cascade is the Vpr gene product. Accordingly, we review the biology of the HIV-1 viral protein R (Vpr) an apoptogenic HIV-1 accessory protein that is packaged into the virus particle. In this review we focus specifically on Vprs ability to induce host cell apoptosis. Recent evidence suggests that Vpr implements a unique mechanism to drive host cell apoptosis, by directly depolarizing the mitochondria membrane potential. Vprs attack on the mitochondria results in release of cytochrome c resulting in activation of the caspase 9 pathway culminating in the activation of caspase 3 and the downstream events of apoptosis. Vpr may interact with the adenine nucleotide translocator (ANT) to prompt this cascade. The role of Vpr-induced apoptosis in HIV pathogenesis is considered.