Role of Occult and Post-acute Phase Replication in Protective Immunity Induced with a Novel Live Attenuated SIV Vaccine.

In order to evaluate the role of persisting virus replication during occult phase immunisation in the live attenuated SIV vaccine model, a novel SIVmac239Δnef variant (SIVrtTA) genetically engineered to replicate in the presence of doxycycline was evaluated for its ability to protect against wild-type SIVmac239. Indian rhesus macaques were vaccinated either with SIVrtTA or with SIVmac239Δnef. Doxycycline was withdrawn from 4 of 8 SIVrtTA vaccinates before challenge with wild-type virus.

Early biodistribution and persistence of a protective live attenuated SIV vaccine elicits localised innate responses in multiple lymphoid tissues.

Vaccination of Mauritian cynomolgus macaques with the attenuated nef-truncated C8 variant of SIVmac251/32H (SIVmacC8) induces early, potent protection against pathogenic, heterologous challenge before the maturation of cognate immunity. To identify processes that contribute to early protection in this model the pathogenesis, anatomical distribution and viral vaccine kinetics were determined in relation to localised innate responses triggered by vaccination. The early biodistribution of SIVmacC8 was defined by rapid, widespread dissemination amongst multiple lymphoid tissues, detectable after 3 days.

Complement-mediated virus infectivity neutralisation by HLA antibodies is associated with sterilising immunity to SIV challenge in the macaque model for HIV/AIDS.

Sterilising immunity is a desired outcome for vaccination against human immunodeficiency virus (HIV) and has been observed in the macaque model using inactivated simian immunodeficiency virus (SIV). This protection was attributed to antibodies specific for cell proteins including human leucocyte antigens (HLA) class I and II incorporated into virions during vaccine and challenge virus preparation. We show here, using HLA bead arrays, that vaccinated macaques protected from virus challenge had higher serum antibody reactivity compared with non-protected animals.

Comment on "Influence of HLA-C expression level on HIV control".

Apps et al. (Reports, 5 April 2013, p. 87) found that high human leukocyte antigen C (HLA-C) expression favors HIV-1 control.

Conditionally-live attenuated SIV upregulates global T effector memory cell frequency under replication permissive conditions.

Background: Live attenuated SIV induces potent protection against superinfection with virulent virus; however the mechanism of this vaccine effect is poorly understood. Such knowledge is important for the development of clinically acceptable vaccine modalities against HIV.

Results: Using a novel, doxycycline dependent, replication-competent live-attenuated SIVmac239Δnef (SIV-rtTAΔnef), we show that under replication-permissive conditions SIV-rtTAΔnef is fully viable.

Transferrin conjugation confers mucosal molecular targeting to a model HIV-1 trimeric gp140 vaccine antigen.

The generation of effective immune responses by mucosal vaccination without the use of inflammatory adjuvants, that compromise the epithelial barrier and recruit new cellular targets, is a key goal of vaccines designed to protect against sexually acquired pathogens. In the present study we use a model HIV antigen (CN54gp140) conjugated to transferrin (Tf) and evaluate the ability of the natural transferrin receptor CD71 to modulate immunity. We show that the conjugated transferrin retained high affinity for its receptor and that the conjugate was specifically transported across an epithelial barrier, co-localizing with MHC Class II(+) cells in the sub-mucosal stroma.

Phase I randomised clinical trial of an HIV-1(CN54), clade C, trimeric envelope vaccine candidate delivered vaginally.

Unlabelled: We conducted a phase 1 double-blind randomised controlled trial (RCT) of a HIV-1 envelope protein (CN54 gp140) candidate vaccine delivered vaginally to assess immunogenicity and safety. It was hypothesised that repeated delivery of gp140 may facilitate antigen uptake and presentation at this mucosal surface. Twenty two healthy female volunteers aged 18-45 years were entered into the trial, the first receiving open-label active product.

Antibody responses after intravaginal immunisation with trimeric HIV-1 CN54 clade C gp140 in Carbopol gel are augmented by systemic priming or boosting with an adjuvanted formulation.

Optimum strategies to elicit and maintain antibodies at mucosal portals of virus entry are critical for the development of vaccines against human immunodeficiency virus (HIV). Here we show in non-human primates that a novel regimen of repeated intravaginal delivery of a non-adjuvanted, soluble recombinant trimeric HIV-1(CN54) clade C envelope glycoprotein (gp140) administered in Carbopol gel can prime for B-cell responses even in the absence of seroconversion. Following 3 cycles of repeated intravaginal administration, throughout each intermenses interval, 3 of 4 macaques produced or boosted systemic and mucosally-detected antibodies upon intramuscular immunisation with gp140 formulated in AS01 adjuvant.

Detection, quantification, and characterisation of HIV/SIV.

Selected techniques for the detection, quantification, and characterisation of HIV1, HIV2, and SIV, as applied to diagnostic and research purposes, are described. Representative nucleic acid testing protocols including nested PCR, RT-PCR, and quantitative real-time PCR, as well as protocols based on virus infectivity, are presented.

Assessment of mucosal immunity to HIV-1.

A key gap in the development and evaluation of HIV-1 vaccines is insufficient knowledge with regard to sampling techniques and assessment of mucosal immune responses required for early prevention and inhibition of viral dissemination. In an attempt to start bridging this gap, the EUROPRISE network of scientists working on HIV-1 vaccine and microbicide research organized a workshop with the aim to review the types of mucosal responses/biomarkers currently measured in mucosal immunology and to define how the mucosal responses/biomarkers are measured and/or the assays and sampling methods used. The Workshop addressed two critical questions: first whether, with current knowledge, it would be possible to define a consensus set of mucosal sampling methods to facilitate cross-species comparisons and ensure standardized implementation in clinical trials; second to determine the remaining challenges (technical and logistical) and their possible solutions for assessing mucosal responses to HIV-1 vaccines.

Vaginal delivery of the recombinant HIV-1 clade-C trimeric gp140 envelope protein CN54gp140 within novel rheologically structured vehicles elicits specific immune responses.

Rheologically structured vehicle (RSV) gels were developed as delivery systems for vaginal mucosal vaccination with an HIV-1 envelope glycoprotein (CN54gp140). RSVs comprised a mucoadhesive matrix-forming and vaginal fluid absorbing polymer. The mucoadhesive and rheological properties of the RSVs were evaluated in vitro, and the distribution, antigenicity and release of CN54gp140 were analysed by ELISA.

Repeated vaginal administration of trimeric HIV-1 clade C gp140 induces serum and mucosal antibody responses.

Vaccine-mediated prevention of primary infection with human immunodeficiency virus (HIV) may require the sustained production of antibody at mucosal portals of entry. Here, we describe a novel approach of repeated mucosal immunization by delivering an HIV-1 envelope glycoprotein (gp) in a gel formulated for intravaginal delivery. Rabbits were immunized over one to three 19-day cycles of intravaginal dosing with soluble recombinant trimeric HIV-1 clade C gp140 administered in Carbopol gel.

Modern mucosal vaccines, adjuvants and microbicides.

Preventing infection at the pathogen portal of entry through induction of mucosal immunity and the use of microbicides has always been an exciting prospect. Moreover, the promise of needle-free prophylaxis is attractive for many reasons. This meeting report highlights some of the critical issues that were discussed concerning recent advances in the field.

Reverse transcriptase inhibitors as potential colorectal microbicides.

We investigated whether reverse transcriptase (RT) inhibitors (RTI) can be combined to inhibit human immunodeficiency virus type 1 (HIV-1) infection of colorectal tissue ex vivo as part of a strategy to develop an effective rectal microbicide. The nucleotide RTI (NRTI) PMPA (tenofovir) and two nonnucleoside RTI (NNRTI), UC-781 and TMC120 (dapivirine), were evaluated. Each compound inhibited the replication of the HIV isolates tested in TZM-bl cells, peripheral blood mononuclear cells, and colorectal explants.

Carriers for the delivery of a vaccine against respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a major cause of bronchiolitis and pneumonia in young children and the elderly. Despite its clinical importance, there is no licensed vaccine available at present. Vaccine development has been hampered by observations of increased pathology after RSV infection in infants vaccinated with formalin-inactivated RSV; incomplete immunity following natural infection; and the need to be effective during the neonatal period when levels of maternal antibody are high.

Macaques infected long-term with attenuated simian immunodeficiency virus (SIVmac) remain resistant to wild-type challenge, despite declining cytotoxic T lymphocyte responses to an immunodominant epitope.

To further investigate mechanisms of protective immunity that are induced by live, attenuated simian immunodeficiency virus (SIV), three macaques were infected with SIVmacGX2, a nef-disrupted molecular clone. In two of these animals, which expressed the MamuA*01 major histocompatibility complex class I allele, loss of functional activity against an SIV-Gag-encoded immunodominant cytotoxic T lymphocyte (CTL) epitope was observed following prolonged infection. Nonetheless, all three animals were resistant to challenge with an uncloned pool of wild-type SIVmac, whereas four naïve controls became infected.

An assessment of different DNA delivery systems for protection against respiratory syncytial virus infection in the murine model: gene-gun delivery induces IgG in the lung.

Immunization with plasmid DNA (pDNA) has the potential to overcome the difficulties of neonatal vaccination that may be required for protection against infection with respiratory syncytial virus (RSV); however, little is known about optimal delivery modalities. In this pilot study we compared mucosal delivery of pDNA encoding RSV F protein encapsulated in poly(DL-lactide-co-glycolide) with delivery of pDNA by gene-gun for the induction of immunity in mice. Intra-gastric or intra-nasal immunization with various doses of microparticles induced weak low levels of RSV-specific serum antibodies in a proportion of mice; in contrast, gene-gun vaccination led to protective immunity associated with a humoral response.

Mucosal immunization with PLGA-microencapsulated DNA primes a SIV-specific CTL response revealed by boosting with cognate recombinant modified vaccinia virus Ankara.

Systemically administered DNA encoding a recombinant human immunodeficiency virus (HIV) derived immunogen effectively primes a cytotoxic T lymphocyte (CTL) response in macaques. In this further pilot study we have evaluated mucosal delivery of DNA as an alternative priming strategy. Plasmid DNA, pTH.

Infection of macaques with simian immunodeficiency virus induces a species-specific antibody response to major histocompatibility complex class I and class II molecules.

Envelopes of retroviruses, including human immunodeficiency virus and simian immunodeficiency virus (SIV), contain host cell proteins that potentially represent novel targets for vaccine development. We show here that sera from rhesus macaques recognized simian major histocompatibility complex (MHC) molecules in response to infection with SIV. Antibodies from these animals did not cross-react with human MHC antigens on mitogen-activated peripheral blood mononuclear cells.

Comparison of the efficacy of early versus late viral proteins in vaccination against SIV.

The immune response against early regulatory proteins of simian- and human immunodeficiency virus (SIV, HIV) has been associated with a milder course of infection. Here, we directly compared vaccination with Tat/Rev versus Pol/Gag. Challenge infection with SIVmac32H (pJ5) suggested that vaccination with Tat/Rev induced cellular immune responses that enabled cynomolgus macaques to more efficiently control SIV replication than the vaccine-induced immune responses against Pol/Gag.

Rhesus macaque resistance to mucosal simian immunodeficiency virus infection is associated with a postentry block in viral replication.

Elucidation of the host factors which influence susceptibility to human immunodeficiency virus or simian immunodeficiency virus (SIV) infection and disease progression has important theoretical and practical implications. Rhesus macaque 359, a vaccine control animal, resisted two successive intravaginal challenges with SIV(mac251) and failed to seroconvert. Here, after an additional intrarectal SIVmac32H challenge, macaque 359 remained highly resistant to infection.

Single oral immunization with replication deficient recombinant adenovirus elicits long-lived transgene-specific cellular and humoral immune responses.

Oral-gastric delivery of vaccines is a preferred route of immunization and is particularly relevant to the development of vaccine-vector systems. We have investigated the ability of a replication deficient (E1-deleted) adenovirus construct (RAd68), which efficiently expresses the measles virus nucleocapsid (N) protein under the control of the strong HCMV IE promoter, to elicit antibody and cytotoxic T cell (CTL) responses in mice following intragastric administration. Measles virus N protein-specific CTL memory and serum antibody responses were analyzed in a total of 140 mice at time points 2-51 weeks after immunization either with a single dose of 10(8) pfu RAd68 or with a fivefold higher dose.