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. Cell-associated viral RNA dynamics identified mesenteric lymph nodes (MLN) and spleen, as well as the gut mucosae, as early major contributors of systemic virus burden. Rapid, localised infection was populated by discrete foci of persisting virus-infected cells. Localised productive infection triggered a broad innate response, with type-1 interferon sensitive IRF-7, STAT-1, TRIM5α and ApoBEC3G genes all upregulated during the acute phase but induction did not prevent viral persistence. Profound changes in vaccine-induced cell-surface markers of immune activation were detected on macrophages, B-cells and dendritic cells (DC-SIGN, S-100, CD40, CD11c, CD123 and CD86). Notably, high DC-SIGN and S100 staining for follicular and interdigitating DCs respectively, in MLN and spleen were detected by 3 days, persisting 20 weeks post-vaccination. Although not formally evaluated, the early biodistribution of SIVmacC8 simultaneously targets multiple lymphoid tissues to induce strong innate immune responses coincident at the same sites critical for early protection from wild-type viruses. HIV vaccines which stimulate appropriate innate, as well as adaptive responses, akin to those generated by live attenuated SIV vaccines, may prove the most efficacious.

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. However, as noted here, HLA-C was assessed with a monoclonal antibody (DT9) that cross-reacts with HLA-E. In the context of the available evidence, this is consistent with the idea that the two leukocyte antigens collaborate to keep the HIV-1 virus at bay.

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.

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. Four categories of RSV vaccine carriers--live-attenuated RSVs, recombinant vectors expressing the protective antigens of RSV, DNA vaccines and subunit vaccines--have been evaluated in animal models and/or clinical trials. So far, studies with live-attenuated virus vaccines highlight the need to improve immunogenicity whilst maintaining a suitable level of attenuation. Studies with recombinant vectors, DNA and subunit vaccines illustrate the pivotal nature of the vaccine carrier in determining the balance between immune-mediated protection against infection and the induction of immune-mediated pulmonary pathology.

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. Tetramer staining revealed the rapid generation of CD8+ T-cell responses against gag- and tat-encoded immunodominant epitopes in MamuA*01+ challenge controls. The dynamics of these T-cell responses to the wild-type virus were similar to those observed following primary infection of the vaccine group with attenuated virus. In contrast, neither tetramer staining nor gamma interferon ELISpot assay revealed an immediate, systemic, anamnestic response in the wild-type-challenged, attenuated SIV-infected animals. Functional CTL capacity had not been lost in this group, as lytic activity was still evident 17 weeks after challenge. Both attenuated and wild-type viruses induced a disseminated CD8+ T-cell response, which was of a higher magnitude in lymphoid tissues than in the periphery. These results suggest that, at least as measured in the periphery, protection against wild-type infection that is induced by live, attenuated SIV is not dependent on a rechallenge-driven expansion of immunodominant epitope-specific CD8+ T cells and, therefore, pre-existing activity may be sufficient to prevent superinfection.

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.HW, encoding a multi-CTL epitope gene, was incorporated into poly(D,L-lactic-co-glycolic acid) microparticles of less than 10 microm in diameter. Five intrarectal immunizations failed to stimulate a circulating vaccine-specific CTL response in 2 Mamu-A*01(+) rhesus macaques. However, 1 week after intradermal immunization with a cognate modified vaccinia virus Ankara vaccine MVA.HW, CTL responses were detected in both animals that persisted until analysis postmortem, 12 weeks after the final boost. In contrast, a weaker and less durable response was seen in an animal vaccinated with the MVA construct alone. Analysis of lymphoid tissues revealed a disseminated CTL response in peripheral and regional lymph nodes but not the spleen of both mucosally primed animals.

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. Vaccination with Tat/Rev resulted in reduced plasma SIV loads compared with control (P=0.058) or Pol/Gag-vaccinated (P=0.089) animals, with undetectable plasma viral loads in two of the four Tat/Rev-vaccinated animals. Therefore, the results warrant further investigation of the early regulatory proteins and their potential for vaccination against HIV.

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. Of the 20 animals analyzed in the first 4-week period following low-dose immunization, 6 mounted low-level splenic CTL responses while 13 animals had CTL in the mesenteric lymph nodes. Splenic CTL responses were largely undetectable at later times. Only 23% of low-dose-immunized mice made serum antibody responses and these were generally of low magnitude and frequently of short duration. In contrast, the majority of animals immunized orally with 5 x 10(8) pfu RAd68 mounted splenic CTL responses (70%) and/or antibody responses (89%). Notably, these responses were stronger and of greater duration than those seen following immunization at the lower dose. Gut mucosal immunization with replication deficient adenoviruses is a promising approach, not only for the development of complementary measles vaccine strategies which may be required for measles virus eradication, but also generally for vaccination against other infections.

Induction of simian immunodeficiency virus (SIV)-specific CTL in rhesus macaques by vaccination with modified vaccinia virus Ankara expressing SIV transgenes: influence of pre-existing anti-vector immunity.

A major aim in AIDS vaccine development is the definition of strategies to stimulate strong and durable cytotoxic T lymphocyte (CTL) responses. Here we report that simian immunodeficiency virus (SIV)-specific CTL developed in 4/4 macaques following a single intramuscular injection of modified vaccinia virus Ankara (MVA) constructs expressing both structural and regulatory/accessory genes of SIV. In two animals Nef-specific responses persisted, but other responses diminished and new responses were not revealed, following further vaccination. Vaccination of another two macaques, expressing Mamu A*01 MHC class I, with MVA constructs containing nef and gag-pol under the control of the moderate strength natural vaccinia virus early/late promoter P7.5, again induced an early Nef-specific response, whereas responses to Gag remained undetectable. Anti-vector immunity induced by this immunization was shown to prevent the efficient stimulation of CTL directed to the cognate Gag epitope, p11C C-M, following vaccination with another MVA construct expressing SIV Gag-Pol under a strong synthetic vaccinia virus-specific promoter. In contrast, vaccination of a previously unexposed animal resulted in a SIV-specific CTL response widely disseminated in lymphoid tissues including lymph nodes associated with the rectal and genital routes of SIV entry. Thus, despite the highly attenuated nature of MVA, repeated immunization may elicit sufficient anti-vector immunity to limit the effectiveness of later vaccination.

Replication-deficient recombinant adenoviruses expressing the human immunodeficiency virus Env antigen can induce both humoral and CTL immune responses in mice.

An effective vaccine against infection with human immunodeficiency virus type 1 (HIV-1) is thought likely to require both a humoral and a CTL immune response. A non-replicating adenovirus vector system has been developed that can induce both a humoral and CTL response to HIV-1 envelope in mice. It is demonstrated that the stimulatory tat/rev 5' splice-donor site sequence is required for efficient expression of HIV-1 env by this adenovirus vector system. rev can be provided bicistronically or in trans to result in good expression of env in vitro. A humoral immune response was detected after two immunizations with a bicistronic recombinant adenovirus (RAd142). The response was dose dependent, 5x10(7) p.f.u. inducing a response in some, but not all, animals and 1x10(8) p.f.u. giving a consistent antibody response. However, CTLs were induced by the lower dose of virus and after only one immunization with the higher dose. A positive CTL response was also seen consistently when the two monocistronic adenoviruses (RAd501 expressing env and RAd46 expressing rev) were given together, although two immunizations were required to give approximately the same level of response as seen with the bicistronic virus. RAd501 on its own also gave a low CTL response when two immunizations were given. It is suggested that a lower level of env expression is required to produce a CTL response than a humoral response and that this nonreplicating adenovirus vector is a good system for inducing CTL.