Reduced Cell-Associated DNA and Improved Viral Control in Macaques following Passive Transfer of a Single Anti-V2 Monoclonal Antibody and Repeated Simian/Human Immunodeficiency Virus Challenges.

A high level of V1V2-specific IgG antibodies (Abs) in vaccinees' sera was the only independent variable that correlated with a reduced risk of human immunodeficiency virus (HIV) acquisition in the RV144 clinical trial. In contrast, IgG avidity, antibody neutralization, and antibody-dependent cellular cytotoxicity each failed as independent correlates of infection. Extended analyses of RV144 samples demonstrated the antiviral activities of V1V2-specific vaccine-induced antibodies. V2-specific antibodies have also been associated with protection from simian immunodeficiency virus (SIV), and the V2i-specific subset of human monoclonal antibodies (MAbs), while poor neutralizers, mediates Fc-dependent antiviral functions in vitro The objective of this study was to determine the protective efficacy of a V2i-specific human MAb, 830A, against mucosal simian/human immunodeficiency virus (SHIV) challenge. V2i MAb binding sites overlap the integrin binding site in the V2 region and are similar to the epitopes bound by antibodies associated with reduced HIV infection rates in RV144. Because the IgG3 subclass was a correlate of reduced infection rates in RV144, we compared passive protection by both IgG1 and IgG3 subclasses of V2i MAb 830A. This experiment represents the first in vivo test of the hypothesis emanating from RV144 and SIV studies that V2i Abs can reduce the risk of infection. The results show that passive transfer with a single V2i MAb, IgG1 830A, reduced plasma and peripheral blood mononuclear cell (PBMC) virus levels and decreased viral DNA in lymphoid tissues compared to controls, but too few animals remained uninfected to achieve significance in reducing the risk of infection. Based on these findings, we conclude that V2i antibodies can impede virus seeding following mucosal challenge, resulting in improved virus control.IMPORTANCE Since the results of the HIV RV144 clinical trial were reported, there has been significant interest in understanding how protection was mediated. Antibodies directed to a subregion of the envelope protein called V1V2 were directly correlated with a reduced risk, and surprisingly low virus neutralization was observed. To determine whether these antibodies alone could mediate protection, we used a human monoclonal antibody directed to V2 with properties similar to those elicited in the vaccine trial for passive infusions in rhesus macaques and challenge with SHIV. The single V2 antibody at the dose given did not significantly reduce the number of infections, but there was a significant reduction in the seeding of virus to the lymph nodes and a decrease in plasma viremia in the HIV antibody-infused macaques compared with the control antibody-infused animals. This finding shows that V2 antibodies mediate antiviral activities in vivo that could contribute to a protective HIV vaccine.

E2 multimeric scaffold for vaccine formulation: immune response by intranasal delivery and transcriptome profile of E2-pulsed dendritic cells.

BACKGROUND: The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses. RESULTS: The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4(+) and CD8(+) cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature. CONCLUSIONS: The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles.

Evaluation of protection induced by a dengue virus serotype 2 envelope domain III protein scaffold/DNA vaccine in non-human primates.

We describe the preclinical development of a dengue virus vaccine targeting the dengue virus serotype 2 (DENV2) envelope domain III (EDIII). This study provides proof-of-principle that a dengue EDIII protein scaffold/DNA vaccine can protect against dengue challenge. The dengue vaccine (EDIII-E2) is composed of both a protein particle and a DNA expression plasmid delivered simultaneously via intramuscular injection (protein) and gene gun (DNA) into rhesus macaques. The protein component can contain a maximum of 60 copies of EDIII presented on a multimeric scaffold of Geobacillus stearothermophilus E2 proteins. The DNA component is composed of the EDIII portion of the envelope gene cloned into an expression plasmid. The EDIII-E2 vaccine elicited robust antibody responses to DENV2, with neutralizing antibody responses detectable following the first boost and reaching titers of greater than 1:100,000 following the second and final boost. Vaccinated and naïve groups of macaques were challenged with DENV2. All vaccinated macaques were protected from detectable viremia by infectious assay, while naïve animals had detectable viremia for 2-7 days post-challenge. All naïve macaques had detectable viral RNA from day 2-10 post-challenge. In the EDIII-E2 group, three macaques were negative for viral RNA and three were found to have detectable viral RNA post challenge. Viremia onset was delayed and the duration was shortened relative to naïve controls. The presence of viral RNA post-challenge corresponded to a 10-30-fold boost in neutralization titers 28 days post challenge, whereas no boost was observed in the fully protected animals. Based on these results, we determine that pre-challenge 50% neutralization titers of >1:6000 correlated with sterilizing protection against DENV2 challenge in EDIII-E2 vaccinated macaques. Identification of the critical correlate of protection for the EDIII-E2 platform in the robust non-human primate model lays the groundwork for further development of a tetravalent EDIII-E2 dengue vaccine.

Multimeric scaffolds displaying the HIV-1 envelope MPER induce MPER-specific antibodies and cross-neutralizing antibodies when co-immunized with gp160 DNA.

Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab) responses toward conserved regions of the viral Envelope (Env). However, the generation of neutralizing Abs (NAbs) targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER) of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.

C1q binding to dengue virus decreases levels of infection and inflammatory molecules transcription in THP-1 cells.

Dengue virus infection elicits a spectrum of clinical presentations ranging from asymptomatic to severe disease. The mechanisms leading to severe dengue are not known, however it has been reported that the complement system is hyper-activated in severe dengue. Screening of complement proteins demonstrated that C1q, a pattern recognition molecule, can bind directly to dengue virus envelope protein and to whole dengue virus serotype 2. Incubation of dengue virus serotype 2 with C1q prior to infection of THP-1 cells led to decreased virus infectivity and modulation of mRNA expression of immunoregulatory molecules suggesting reduced inflammatory responses.

Association between magnitude of the virus-specific plasmablast response and disease severity in dengue patients.

Dengue is a globally expanding disease caused by infection with dengue virus (DENV) that ranges from febrile illness to acute disease with serious complications. Secondary infection predisposes individuals to more severe disease, and B lymphocytes may play a role in this phenomenon through production of Ab that enhance infection. To better define the acute B cell response during dengue, we analyzed peripheral B cells from an adult Brazilian hospital cohort with primary and secondary DENV infections of varying clinical severity. Circulating B cells in dengue patients were proliferating, activated, and apoptotic relative to individuals with other febrile illnesses. Severe secondary DENV infection was associated with extraordinary peak plasmablast frequencies between 4 and 7 d of illness, averaging 46% and reaching 87% of B cells, significantly greater than those seen in mild illness or primary infections. On average >70% of IgG-secreting cells in individuals with severe secondary DENV infection were DENV specific. Plasmablasts produced Ab that cross-reacted with heterotypic DENV serotypes, but with a 3-fold greater reactivity to DENV-3, the infecting serotype. Plasmablast frequency did not correlate with acute serum-neutralizing Ab titers to any DENV serotype regardless of severity of disease. These findings indicate that massive expansion of DENV-specific and serotype cross-reactive plasmablasts occurs in acute secondary DENV infection of adults in Brazil, which is associated with increasing disease severity.

Evaluation of heterologous vaginal SHIV SF162p4 infection following vaccination with a polyvalent Clade B virus-like particle vaccine.

The vast diversity of HIV-1 infections has greatly impeded the development of a successful HIV-1/AIDS vaccine. Previous vaccine work has demonstrated limited levels of protection against SHIV/SIV infection, but protection was observed only when the challenge virus was directly matched to the vaccine strain. As it is likely impossible to directly match the vaccine strain to all infecting strains in nature, it is necessary to develop an HIV-1 vaccine that can protect against a heterologous viral challenge. In this study we investigated the ability of polyvalent and consensus vaccines to protect against a heterologous clade B challenge. Rhesus macaques were vaccinated with ConB or PolyB virus-like particle vaccines. All vaccines were highly immunogenic with high titers of antibody found in all vaccinated groups against SIV Gag. Antibody responses were also observed against a diverse panel of clade B envelopes. Following vaccination nonhuman primates (NHPs) were challenged via the vaginal route with SHIV(SF162p4). The PolyB vaccine induced a 66.7% reduction in the rate of infection as well as causing a two log reduction in viral burden if infection was not blocked. ConB vaccination had no effect on either the infection rate or viral burden. These results indicate that a polyvalent clade-matched vaccine is better able to protect against a heterologous challenge as compared to a consensus vaccine.

Human immunodeficiency virus-like particles with consensus envelopes elicited broader cell-mediated peripheral and mucosal immune responses than polyvalent and monovalent Env vaccines.

Envelope (Env) sequences from human immunodeficiency virus (HIV) strains can vary by 15-20% within a single clade and as much as 35% between clades. Previous AIDS vaccines based upon a single isolate often could not elicit protective immune responses against heterologous viral challenges. In order to address the vast sequence diversity in Env sequences, consensus sequences were constructed for clade B and clade C envelopes and delivered to the mouse lung mucosa on the surface of virus-like particles (VLP). Consensus sequences decrease the genetic difference between the vaccine strain and any given viral isolate. The elicited immune responses were compared to a mixture of VLPs with Envs from primary viral isolates. This polyvalent vaccine approach contains multiple, diverse Envs to increase the breadth of epitopes recognized by the immune response and thereby increase the potential number of primary isolates recognized. Both consensus and polyvalent clade B Env VLP vaccines elicited cell-mediated immune responses that recognized a broader number of clade B Env peptides than a control monovalent Env VLP vaccine in both the systemic and the mucosal immune compartments. All three clade C Env vaccine strategies elicited similar responses to clade C peptides. However, both the consensus B and C Env VLP vaccines were more effective at eliciting cross-reactive cellular immune responses to epitopes in other clades. This is the first study to directly compare the breadth of cell-mediated immune responses elicited by consensus and polyvalent Env vaccines.

Viral sequence diversity: challenges for AIDS vaccine designs.

Among the greatest challenges facing AIDS vaccine development is the intrinsic diversity among circulating populations of HIV-1 in various geographical locations and the need to develop vaccines that can elicit enduring protective immunity to variant HIV-1 strains. While variation is observed in all of the viral proteins, the greatest diversity is localized to the viral envelope glycoproteins, evidently reflecting the predominant role of these proteins in eliciting host immune recognition and responses that result in progressive evolution of the envelope proteins during persistent infection. Interestingly, while envelope glycoprotein variation is widely assumed to be a major obstacle to AIDS vaccine development, there is very little experimental data in animal or human lentivirus systems addressing this critical issue. In this review, the state of vaccine development to address envelope diversity will be presented, focusing on the use of centralized and polyvalent sequence design as mechanisms to elicit broadly reactive immune responses.

Developing broadly reactive HIV-1/AIDS vaccines: a review of polyvalent and centralized HIV-1 vaccines.

The development of an HIV/AIDS vaccine requires consideration of the large diversity of viral isolates. In 2005, there were 5 million new cases of HIV infection and over 4 million deaths due to AIDS. An HIV vaccine is needed to prevent the spread of this virus. One of the greatest challenges to developing a preventative HIV vaccine is the diversity of HIV-1 isolates. Env sequences can differ by as much as 35% between isolates from different clades and by as much as 10% within a clade. Two main strategies to address viral diversity for HIV vaccine development are the use of polymeric- or centralized-based immunogens. Polymeric-based vaccines, which have been used for polio and pneumococcus vaccines, use components from a range of viral isolates to increase the breadth of immune recognition. Centralized sequences decrease the sequence diversity by encoding the most common amino acid at each position from a diverse pool of viral isolates. These sequences are derived using the consensus, center-of-the-tree, or ancestral methods. The use of polyvalent- and centralized-based vaccines induce broadly reactive immune responses, however it is unclear whether the use of these sequences will increase protection against diverse HIV-1 infection. This review will summarize the current uses of polyvalent and centralized vaccines to increase immune breadth that may determine future directions for HIV-1 vaccine development.

Membrane embedded HIV-1 envelope on the surface of a virus-like particle elicits broader immune responses than soluble envelopes.

Virally regulated HIV-1 particles were expressed from DNA plasmids encoding Gag, protease, reverse transcriptase, Vpu, Tat, Rev, and Env. The sequences for integrase, Vpr, Vif, Nef, and the long terminal repeats (LTRs) were deleted. Mutations were engineered into the VLP genome to produce particles deficient in activities associated with viral reverse transcriptase, RNase H, and RNA packaging. Each plasmid efficiently secreted particles from primate cells in vitro and particles were purified from the supernatants and used as immunogens. Mice (BALB/c) were vaccinated intranasally (day 1 and weeks 3 and 6) with purified VLPs and the elicited immunity was compared to particles without Env (Gag(p55)), to soluble monomeric Env(gp120), or to soluble trimerized Env(gp140). Only mice vaccinated with VLPs had robust anti-Env cellular immunity. In contrast, all mice had high titer anti-Env serum antibody (IgG). However, VLP-vaccinated mice had antisera that detected a broader number of linear Env peptides, had anti-Env mucosal IgA and IgG, as well as higher titers of serum neutralizing antibodies. VLPs elicited high titer antibodies that recognized linear regions in V4-C5 and the ectodomain of gp41, but did not recognize V3. These lentiviral VLPs are effective mucosal immunogens that elicit broader immunity against Env determinants in both the systemic and mucosal immune compartments than soluble forms of Env.

Lentivirus-like particles without reverse transcriptase elicit efficient immune responses.

Following infection by HIV or SIV, reverse transcriptase (RT) directs the conversion of the single-stranded RNA genome into a double-stranded DNA molecule that integrates into the host cell genome. RT encodes for several immunogenic epitopes that are desirable for inclusion in a human vaccine for HIV infection, however, issues of safety have dampened enthusiasm for inclusion of an enzymatically-active RT molecule into an AIDS vaccine. In this study, virally-regulated, replication-incompetent lentiviral particles were expressed from DNA plasmids. The sequences for integrase, Vpr, Vif, Nef, and the long terminal repeats (LTRs) were deleted and mutations were engineered into capsid to decreases RNA packaging. Virus-like particles incorporated no RT (HIV-VLP DeltaRT or SHIV-VLP DeltaRT) or contained a full-length enzymatically-inactivated RT molecule (HIV-VLP or SHIV-VLP). Each secreted VLP was enveloped with a lipid bilayer derived from primate cells with embedded, native viral envelopes in similar concentrations as infectious virions. BALB/c mice were vaccinated (weeks 0, 3, and 6) with purified VLPs via intranasal inoculation in the presence of cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs). All VLPs, with or without RT, elicited both robust humoral and cellular immune responses to Gag, Pol, and Env antigens. Therefore, the lack of RT enhances the safety of these VLPs for use in future human clinical trials without a significant reduction in the overall immunogenicity of these VLP immunogens.

Virus-like particles: designing an effective AIDS vaccine.

Viruses that infect eukaryotic organisms have the unique characteristic of self-assembling into particles. The mammalian immune system is highly attuned to recognizing and attacking these viral particles following infection. The use of particle-based immunogens, often delivered as live-attenuated viruses, has been an effective vaccination strategy for a variety of viruses. The development of an effective vaccine against the human immunodeficiency virus (HIV) has proven to be a challenge, since HIV infects cells of the immune system causing severe immunodeficiency resulting in the syndrome known as AIDS. In addition, the ability of the virus to adapt to immune pressure and reside in an integrated form in host cells presents hurdles for vaccinologists to overcome. A particle-based vaccine strategy has promise for eliciting high titer, long-lived, immune responses to a diverse number of viral epitopes against different HIV antigens. Live-attenuated viruses are effective at generating both cellular and humoral immune responses. However, while these vaccines stimulate immunity, challenged animals rarely clear the viral infection and the degree of attenuation directly correlates with protection from disease. Further, a live-attenuated vaccine has the potential to revert to a pathogenic form. Alternatively, virus-like particles (VLPs) mimic the viral particle without causing an immunodeficiency disease. VLPs are self-assembling, non-replicating, non-pathogenic particles that are similar in size and conformation to intact virions. A variety of VLPs for lentiviruses are currently in preclinical and clinical trials. This review focuses on our current status of VLP-based AIDS vaccines, regarding issues of purification and immune design for animal and clinical trials.