Characterization of new anti-IL-6 antibodies revealed high potency candidates for intracellular cytokine detection and specific targeting of IL-6 receptor binding sites.

Interleukin-6 (IL-6) expression and secretion, induced by inflammatory processes, stimulate the acute phase response cascade. The overexpression of IL-6 contributes to a variety of inflammatory diseases, e.g. rheumatoid arthritis, Castleman's disease, multiple myeloma, and prostate cancer. Screening for high amounts of IL-6 in the patients' blood serum can be crucial for an adequate treatment. In this study, five novel murine monoclonal antibodies (mAbs) reactive to human IL-6 were generated. The mAbs were characterized for potential diagnostic purposes and recombinant antibodies were derived thereof. Initial epitope mapping using a combination of blocking experiments and Hyper-IL-6, a fusion protein consisting of IL-6 and the soluble IL-6 receptor revealed distinct but overlapping binding sites. At least one of the mAbs was found to interact with the region of IL-6/ IL-R complex formation. Three mAbs were applied successfully in intracellular staining by flow cytometry, whereas one of the mAbs showed comparable binding as a reference reagent. Furthermore, the mAbs were tested for applications in various immunological assays such as ELISA, Western blot and surface plasmon resonance spectroscopy (SPR), using IL-6 from commercial sources as well as in-house produced protein (IL-6_IME). The limit of detection was determined by sandwich ELISA (0.5 ng/mL, SD ±0.005). Our results also demonstrated that the recombinant IL-6 produced was functional and correctly folded. These findings support the use of the generated mAb clones as promising candidates for application in various immunological assays for diagnostic and scientific purposes.

Rhesus immune responses to SIV Gag expressed by recombinant BCG vectors are independent from pre-existing mycobacterial immunity.

BACKGROUND: A recombinant Mycobacterium bovis BCG (rBCG) vector expressing HIV transgenes is an attractive candidate as a dual vaccine against HIV and TB. However, pre-existing immune responses to mycobacteria may influence immune responses to rBCG. We analyzed data from a rhesus rBCG trial to determine the effect of pre-existing mycobacterial immune responses on the vaccine-induced responses to the vector and expressed transgene. METHODS: Indian-origin rhesus macaques were primed with rBCG expressing simian immunodeficiency virus (SIV) Gag and boosted with attenuated vaccinia NYVAC gag-pol. Mycobacteria responses were measured by Mycobacterium tuberculosis (Mtb) purified protein derivative (PPD) interferon-γ ELISpot and Mtb whole cell lysate (WCL) ELISA. SIV Gag responses were measured by SIV Gag ELISpot and by p11C tetramer binding. RESULTS: Baseline Mtb PPD ELISpot responses and Mtb WCL antibody responses in rhesus macaques overlapped those in human populations. Cellular and antibody responses boosted sharply 4 weeks after rBCG vaccination. Mtb WCL antibody titers at 4 weeks correlated with baseline titers. Primates vaccinated with rBCG developed strong SIV Gag ELISpot and p11C tetramer responses after rBCG prime and NYVAC boost. There were no correlations between the pre-existing mycobacterial immune responses and the SIV Gag T cell responses after vaccination. CONCLUSIONS: Rhesus immune responses to SIV Gag expressed by rBCG vectors were independent from pre-existing anti-mycobacterial immunity. Rhesus macaques may serve as a surrogate for investigations of pre-existing anti-mycobacterial immunity in humans.

Improving Mycobacterium bovis bacillus Calmette-Guèrin as a vaccine delivery vector for viral antigens by incorporation of glycolipid activators of NKT cells.

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.

Recombinant Mycobacterium bovis bacillus Calmette-Guérin vectors prime for strong cellular responses to simian immunodeficiency virus gag in rhesus macaques.

Live attenuated nonpathogenic Mycobacterium bovis bacillus Calmette-Guérin (BCG) mediates long-lasting immune responses, has been safely administered as a tuberculosis vaccine to billions of humans, and is affordable to produce as a vaccine vector. These characteristics make it very attractive as a human immunodeficiency virus (HIV) vaccine vector candidate. Here, we assessed the immunogenicity of recombinant BCG (rBCG) constructs with different simian immunodeficiency virus (SIV)gag expression cassettes as priming agents followed by a recombinant replication-incompetent New York vaccinia virus (NYVAC) boost in rhesus macaques. Unmutated rBCG constructs were used in comparison to mutants with gene deletions identified in an in vitro screen for augmented immunogenicity. We demonstrated that BCG-SIVgag is able to elicit robust transgene-specific priming responses, resulting in strong SIV epitope-specific cellular immune responses. While enhanced immunogenicity was sustained at moderate levels for >1 year following the heterologous boost vaccination, we were unable to demonstrate a protective effect after repeated rectal mucosal challenges with pathogenic SIVmac251. Our findings highlight the potential for rBCG vaccines to stimulate effective cross-priming and enhanced major histocompatibility complex class I presentation, suggesting that combining this approach with other immunogens may contribute to the development of effective vaccine regimens against HIV.

The selection of low envelope glycoprotein reactivity to soluble CD4 and cold during simian-human immunodeficiency virus infection of rhesus macaques.

Envelope glycoprotein (Env) reactivity (ER) describes the propensity of human immunodeficiency virus type 1 (HIV-1) Env to change conformation from the metastable unliganded state in response to the binding of ligands (antibodies and soluble CD4 [sCD4]) or incubation in the cold. To investigate Env properties that favor in vivo persistence, we inoculated rhesus macaques with three closely related CCR5-tropic simian-human immunodeficiency viruses (SHIVs) that differ in ER to cold (ERcold) and ER to sCD4 (ERsCD4); these SHIVs were neutralized by antibodies equivalently and thus were similar in ERantibody. All three SHIVs achieved high levels of acute viremia in the monkeys without alteration of their Env sequences, indicating that neither ERcold nor ERsCD4 significantly influences the establishment of infection. Between 14 and 100 days following infection, viruses with high ERcold and ERsCD4 were counterselected. Remarkably, the virus variant with low ERcold and low ERsCD4 did not elicit a neutralizing antibody response against the infecting virus, despite the generation of high levels of anti-Env antibodies in the infected monkeys. All viruses that achieved persistent viremia escaped from any autologous neutralizing antibodies and exhibited low ERcold and low ERsCD4. One set of gp120 changes determined the decrease in ERcold and ERsCD4, and a different set of gp120 changes determined resistance to autologous neutralizing antibodies. Each set of changes contributed to a reduction in Env-mediated entry. During infection of monkeys, any Env replication fitness costs associated with decreases in ERcold and ERsCD4 may be offset by minimizing the elicitation of autologous neutralizing antibodies.

Heterologous prime/boost immunizations of rhesus monkeys using chimpanzee adenovirus vectors.

Pre-existing immunity to human adenovirus serotype 5 (AdHu5) has been shown to suppress the immunogenicity of recombinant Ad5 (rAdHu5) vector-based vaccines for human immunodeficiency virus type 1 (HIV-1) in both preclinical studies and clinical trials. As a potential solution to this problem we developed adenovirus vaccine vectors of chimpanzee origin. In the present study we assessed the immunogenicity of various chimpanzee adenovirus vectors in a prime/boost regimen to HIV-1 envelope and SIV Gag-Pol in rhesus monkeys and their ability to protect against pathogenic viral challenge. Although rAdHu5-primed monkeys had higher magnitude T cell responses than rAdC7 or rAdC68 prior to challenge, the rAdC7-rAdC1/C5 and rAdHu5-rAdC1/C5 immunizations resulted in comparable magnitude recall cellular immune responses and comparable level of control of viremia post-challenge.

Recombinant Mycobacterium bovis BCG prime-recombinant adenovirus boost vaccination in rhesus monkeys elicits robust polyfunctional simian immunodeficiency virus-specific T-cell responses.

While mycobacteria have been proposed as vaccine vectors because of their persistence and safety, little has been done systematically to optimize their immunogenicity in nonhuman primates. We successfully generated recombinant Mycobacterium bovis BCG (rBCG) expressing simian immunodeficiency virus (SIV) Gag and Pol as multigenic, nonintegrating vectors, but rBCG-expressing SIV Env was unstable. A dose and route determination study in rhesus monkeys revealed that intramuscular administration of rBCG was associated with local reactogenicity, whereas intravenous and intradermal administration of 10(6) to 10(8) CFU of rBCG was well tolerated. After single or repeat rBCG inoculations, monkeys developed high-frequency gamma interferon enzyme-linked immunospot responses against BCG purified protein derivative. However, the same animals developed only modest SIV-specific CD8(+) T-cell responses. Nevertheless, high-frequency SIV-specific cellular responses were observed in the rBCG-primed monkeys after boosting with recombinant adenovirus 5 (rAd5) expressing the SIV antigens. These cellular responses were of greater magnitude and more persistent than those generated after vaccination with rAd5 alone. The vaccine-elicited cellular responses were predominantly polyfunctional CD8(+) T cells. These findings support the further exploration of mycobacteria as priming vaccine vectors.

Simian immunodeficiency virus (SIV)-specific CD8+ T-cell responses in vervet African green monkeys chronically infected with SIVagm.

African green monkeys (AGM) do not develop overt signs of disease following simian immunodeficiency virus (SIV) infection. While it is still unknown how natural hosts like AGM can cope with this lentivirus infection, a large number of investigations have shown that CD8(+) T-cell responses are critical for the containment of AIDS viruses in humans and Asian nonhuman primates. Here we have compared the phenotypes of T-cell subsets and magnitudes of SIV-specific CD8(+) T-cell responses in vervet AGM chronically infected with SIVagm and rhesus monkeys (RM) infected with SIVmac. In comparison to RM, vervet AGM exhibited weaker signs of immune activation and associated proliferation of CD8(+) T cells as detected by granzyme B, Ki-67, and programmed death 1 staining. By gamma interferon enzyme-linked immunospot assay and intracellular cytokine staining, SIV Gag- and Env-specific immune responses were detectable at variable but lower levels in vervet AGM than in RM. These observations demonstrate that natural hosts like SIV-infected vervet AGM develop SIV-specific T-cell responses, but the disease-free course of infection does not depend on the generation of robust CD8(+) T-cell responses.

The efficacy of T cell-mediated immune responses is reduced by the envelope protein of the chimeric HIV-1/SIV-KB9 virus in vivo.

Gp120 is a critical component of the envelope of HIV-1. Its role in viral entry is well described. In view of its position on the viral envelope, gp120 is a part of the retrovirus that immune cells encounter first and has the potential to influence antiretroviral immune responses. We propose that high levels of gp120 are present in tissues and may contribute to the failure of the immune system to fully control and ultimately clear the virus. Herein, we show for the first time that lymphoid tissues from acutely HIV-1/SIV (SHIV)-KB9-infected macaques contain deposits of gp120 at concentrations that are high enough to induce suppressive effects on T cells, thus negatively regulating the antiviral CTL response and contributing to virus survival and persistence. We also demonstrate that SHIV-KB9 gp120 influences functional T cell responses during SHIV infection in a manner that suppresses degranulation and cytokine secretion by CTLs. Finally, we show that regulatory T cells accumulate in lymphoid tissues during acute infection and that they respond to gp120 by producing TGFbeta, a known suppressant of cytotoxic T cell activity. These findings have significant implications for our understanding of the contribution of non-entry-related functions of HIV-1 gp120 to the pathogenesis of HIV/AIDS.

Effect of preexisting immunity to adenovirus human serotype 5 antigens on the immune responses of nonhuman primates to vaccine regimens based on human- or chimpanzee-derived adenovirus vectors.

In this study we compared a prime-boost regimen with two serologically distinct replication-defective adenovirus (Ad) vectors derived from chimpanzee serotypes C68 and C1 expressing Gag, Pol, gp140, and Nef of human immunodeficiency virus type 1 with a regimen in which replication-defective Ad vectors of the human serotype 5 (AdHu5) were given twice. Experiments were conducted in rhesus macaques that had or had not been preexposed to antigens of AdHu5. There was no significant difference in T-cell responses tested from peripheral blood of the different groups, although responses were overall highest in nonpreexposed animals immunized with the chimpanzee Ad vectors. Preexisting immunity to AdHu5 completely inhibited induction of transgene product-specific antibodies by the AdHu5 vectors without affecting antibody responses to the chimpanzee vectors. Upon euthanasia, T-cell responses were tested from a number of tissues. Preexisting immunity to AdHu5, commonly found in humans, changed the homing pattern of vaccine-induced T cells. In AdHu5-preexposed animals vaccinated with the chimpanzee Ad vectors, frequencies of transgene-specific T cells were higher in spleens than in blood, and in most preexposed animals vaccinated either with AdHu5 vectors or chimpanzee adenovirus vectors, frequencies of such T cells were exceptionally high in livers. The latter results indicate that analysis of T-cell responses solely from blood mononuclear cells of vaccine recipients may not suffice to compare the potencies of different vaccine regimens.

A replication-competent adenovirus-human immunodeficiency virus (Ad-HIV) tat and Ad-HIV env priming/Tat and envelope protein boosting regimen elicits enhanced protective efficacy against simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques.

We previously demonstrated that replication-competent adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinant prime/protein boost regimens elicit potent immunogenicity and strong, durable protection of rhesus macaques against SIV(mac251). Additionally, native Tat vaccines have conferred strong protection against simian/human immunodeficiency virus SHIV(89.6P) challenge of cynomolgus monkeys, while native, inactivated, or vectored Tat vaccines have failed to elicit similar protective efficacy in rhesus macaques. Here we asked if priming rhesus macaques with replicating Ad-human immunodeficiency virus (HIV) tat and boosting with the Tat protein would elicit protection against SHIV(89.6P). We also evaluated a Tat/Env regimen, adding an Ad-HIV env recombinant and envelope protein boost to test whether envelope antibodies would augment acute-phase protection. Further, expecting cellular immunity to enhance chronic viremia control, we tested a multigenic group: Ad-HIV tat, -HIV env, -SIV gag, and -SIV nef recombinants and Tat, Env, and Nef proteins. All regimens were immunogenic. A hierarchy was observed in enzyme-linked immunospot responses (with the strongest response for Env, followed by Gag, followed by Nef, followed by Tat) and antibody titers (with the highest titer for Env, followed by Tat, followed by Nef, followed by Gag). Following intravenous SHIV(89.6P) challenge, all macaques became infected. Compared to controls, no protection was seen in the Tat-only group, confirming previous reports for rhesus macaques. However, the multigenic group blunted acute viremia by approximately 1 log (P = 0.017), and both the multigenic and Tat/Env groups reduced chronic viremia by 3 and 4 logs, respectively, compared to controls (multigenic, P = 0.0003; Tat/Env, P < 0.0001). The strikingly greater reduction in the Tat/Env group than in the multigenic group (P = 0.014) was correlated with Tat and Env binding antibodies. Since prechallenge anti-Env antibodies lacked SHIV(89.6P)-neutralizing activity, other functional anti-Env and anti-Tat activities are under investigation, as is a possible synergy between the Tat and Env immunogens.

A human T-cell leukemia virus type 1 regulatory element enhances the immunogenicity of human immunodeficiency virus type 1 DNA vaccines in mice and nonhuman primates.

Plasmid DNA vaccines elicit potent and protective immune responses in numerous small-animal models of infectious diseases. However, their immunogenicity in primates appears less potent. Here we investigate a novel approach that optimizes regulatory elements in the plasmid backbone to improve the immunogenicity of DNA vaccines. Among various regions analyzed, we found that the addition of a regulatory sequence from the R region of the long terminal repeat from human T-cell leukemia virus type 1 (HTLV-1) to the cytomegalovirus (CMV) enhancer/promoter increased transgene expression 5- to 10-fold and improved cellular immune responses to human immunodeficiency virus type 1 (HIV-1) antigens. In cynomolgus monkeys, DNA vaccines containing the CMV enhancer/promoter with the HTLV-1 R region (CMV/R) induced markedly higher cellular immune responses to HIV-1 Env from clades A, B, and C and to HIV-1 Gag-Pol-Nef compared with the parental DNA vaccines. These data demonstrate that optimization of specific regulatory elements can substantially improve the immunogenicity of DNA vaccines encoding multiple antigens in small animals and in nonhuman primates. This strategy could therefore be explored as a potential method to enhance DNA vaccine immunogenicity in humans.

Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys.

Because a strategy to elicit broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies has not yet been found, the role of an Env immunogen in HIV-1 vaccine candidates remains undefined. We sought to determine whether an HIV-1 Env immunogen genetically disparate from the Env of the challenge virus can contribute to protective immunity. We vaccinated Indian-origin rhesus monkeys with Gag-Pol-Nef immunogens, alone or in combination with Env immunogens that were either matched or mismatched with the challenge virus. These animals were then challenged with a pathogenic simian-human immunodeficiency virus. The vaccine regimen included a plasmid DNA prime and replication-defective adenoviral vector boost. Vaccine regimens that included the matched or mismatched Env immunogens conferred better protection against CD4(+) T-lymphocyte loss than that seen with comparable regimens that did not include Env immunogens. This increment in protective immunity was associated with anamnestic Env-specific cellular immunity that developed in the early days following viral challenge. These data suggest that T-lymphocyte immunity to Env can broaden the protective cellular immune response to HIV despite significant sequence diversity of the strains of the Env immunogens and can contribute to immune protection in this AIDS vaccine model.

Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-Ad5 immunity.

The high prevalence of pre-existing immunity to adenovirus serotype 5 (Ad5) in human populations may substantially limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for HIV-1 and other pathogens. A potential solution to this problem is to use vaccine vectors derived from adenovirus (Ad) serotypes that are rare in humans, such as Ad35. However, cross-reactive immune responses between heterologous Ad serotypes have been described and could prove a major limitation of this strategy. In particular, the extent of immunologic cross-reactivity between Ad5 and Ad35 has not previously been determined. In this study we investigate the impact of pre-existing anti-Ad5 immunity on the immunogenicity of candidate rAd5 and rAd35 vaccines expressing SIV Gag in mice. Anti-Ad5 immunity at levels typically found in humans dramatically blunted the immunogenicity of rAd5-Gag. In contrast, even high levels of anti-Ad5 immunity did not substantially suppress Gag-specific cellular immune responses elicited by rAd35-Gag. Low levels of cross-reactive Ad5/Ad35-specific CD4(+) T lymphocyte responses were observed, but were insufficient to suppress vaccine immunogenicity. These data demonstrate the potential utility of Ad35 as a candidate vaccine vector that is minimally suppressed by anti-Ad5 immunity. Moreover, these studies suggest that using Ad vectors derived from immunologically distinct serotypes may be an effective and general strategy to overcome the suppressive effects of pre-existing anti-Ad immunity.