CCL19 as an adjuvant for intradermal gene gun immunization in a Her2/neu mouse tumor model: improved vaccine efficacy and a role for B cells as APC.

The aim of this study was to evaluate the efficacy of the chemokine CCL19 (ELC) as an adjuvant for intradermal gene gun delivery of Her2/neu DNA and to investigate the role of B cells in CCL19-mediated enhancement of immune responses. Balb/c mice were immunized intramuscularly (i.m.) on days 1 and 15 with plasmid DNA (pDNA) (100 µg DNA) or intradermally (i.d.) by gene gun delivery (1-2 µg DNA). Administration of pDNA encoding Her2/neu (pDNA(Her2/neu) was compared with pDNA(Her2/neu) plus pDNA(CCL19), pDNA(CCL19), mock vector or uncoated gold particles/phosphate-buffered saline (PBS). Tumor challenge was performed subcutaneously on day 25 with syngeneic Her2/neu(+) tumor cells (D2F2/E2). Intradermal immunization by gene gun led to an enhancement of tumor protection by the DNA vaccine as compared with i.m. immunization. The protective effect of the vaccine was further enhanced by coadministration of pDNA(CCL19) both after i.m. and i.d. immunization. Tumor protection was associated with Her2/neu-specific T cell and humoral immune responses. Experiments in B-cell-deficient µMT mice showed that B cells are crucial for CCL19-mediated enhancement of tumor rejection, most likely as antigen-presenting B cells. DNA vaccines against Her2/neu may play a future role in the treatment of Her2/neu-positive breast cancer patients in a clinical situation of minimal residual disease.

Codon-optimization of the hemagglutinin gene from the novel swine origin H1N1 influenza virus has differential effects on CD4(+) T-cell responses and immune effector mechanisms following DNA electroporation in mice.

DNA electroporation is a powerful vaccine strategy that could be rapidly adapted to address emerging viruses. We therefore compared cellular and humoral immune responses in mice vaccinated with DNA expression plasmids encoding either the wildtype or a codon-optimized sequence of hemagglutinin from the novel swine origin H1N1 influenza virus. While expression of HA from the wildtype sequence was hardly detectable, the H1N1 hemagglutinin was well expressed from the codon-optimized sequence. Despite poor expression of the wildtype sequence, both plasmids induced similar levels of CD4(+) T-cell responses. However, CD8(+) T-cell and antibody responses were substantially higher after immunization with the codon-optimized DNA vaccine. Thus, efficient induction of immune effector mechanisms against HA of the novel H1N1 influenza virus requires codon-optimization of the DNA vaccines. Since DNA vaccines and several viral vector vaccines employ the same cellular RNA-Polymerase II dependent expression pathway, the poor expression levels from wildtype HA sequences might also limit the induction of immune effector mechanisms by such viral vector vaccines.

A neutralization assay for HIV-2 based on measurement of provirus integration by duplex real-time PCR.

Specific, effective and rapid neutralization assays are crucial for the development of an HIV vaccine based on the stimulation of neutralizing antibodies and the development of such an assay for the human immunodeficiency virus-2 (HIV-2) is described. Virus neutralization was measured as the reduction of provirus integration using a duplex real-time PCR with high efficiency (99.4%). This PCR uses primers and a probe specific for the proviral LTR. Amplification and quantitative analysis of the cellular GAPDH gene was carried out in parallel to control for toxic or growth-inhibitory components in the sera. The neutralization assay was used to screen sera from 23 HIV-2 infected patients. 21 sera were able to neutralize HIV-2(60415K), 20 sera neutralized HIV-2(7312A) and 7 sera cross-neutralized HIV-1 IIIB. In contrast, when 14 of these sera were tested in parallel with a conventional neutralization assay based on a p27Gag capture ELISA, only one was found to neutralize HIV-2(60415K) and 11 to neutralize HIV-2(7312A) compared with 12 and 13 sera respectively using the PCR-based assay.

Preparation and characterization of new challenge stocks of SIVmac32H J5 following rapid serial passage of virus in vivo.

BACKGROUND: A new challenge stock of the simian immunodeficiency virus SIVmacJ5 has been produced following passage in vivo. METHODS: SIVmacJ5 3/92 (J5M), was passaged serially through cynomolgus macaques (Macaca fascicularis) by intravenous inoculation of infected spleen cells isolated and prepared 14 days post-infection. Two challenge stocks, SIVmacJ5 S61MLN and SIVmacJ5 S62spl, were prepared by culture of lymphoid tissue ex vivo. RESULTS: These virus stocks appeared better adapted for replication in M. fascicularis as demonstrated by a greater persistence of recoverable live virus from the periphery and increased pathology in lymphoid tissues 20 weeks post-challenge as detected by immunohistochemistry. Sequence analysis of the envelope gene from these stocks did not identify marked diversification of sequence as a result of this procedure. CONCLUSIONS: These stocks display more robust peripheral persistence and tissue pathology in cynomolgus macaques and should prove valuable analysing recombinant vaccines based upon SIVmacJ5 transgenes.

Analysis of SIV-specific CTL in the rhesus macaque model of AIDS: the use of simian fibroblasts as an alternative source of target cells for chromium release assays.

The simian immunodeficiency virus (SIV) model of AIDS is widely used for the development of human immunodeficiency virus (HIV) vaccine strategies, particularly for the analysis of correlates of protective immunity. As it is not always possible to establish autologous B-lymphoblastoid cell lines (B-LCL) for use as targets in the analysis of cytotoxic T cell (CTL) activity, we have compared B-LCL with primary simian skin cells. Using a well-defined SIV gag-encoded CTL epitope restricted by Mamu A*01 major histocompatibility complex (MHC) class I, we have shown that peripheral blood mononuclear cells (PBMC) from vaccinated and infected macaques can kill MHC class I-matched skin fibroblasts presenting the cognate epitope but that skin fibroblasts are a less sensitive target than B-LCL for the detection of CTL.

Effect of vaccination with recombinant modified vaccinia virus Ankara expressing structural and regulatory genes of SIV(macJ5) on the kinetics of SIV replication in cynomolgus monkeys.

The efficacy of a multicomponent vaccination with modified vaccinia Ankara constructs (rMVA) expressing structural and regulatory genes of simian immunodeficiency virus (SIV(mac251/32H/J5)) was investigated in cynomolgus monkeys, following challenge with a pathogenic SIV. Vaccination with rMVA-J5 performed at week 0, 12, and 24 induced a moderate proliferative response to whole SIV, a detectable humoral response to all but Nef SIV antigens, and failed to induce neutralizing antibodies. Two months after the last boost, the monkeys were challenged intravenously with 50 MID50 of SIV(mac251). All control monkeys, previously inoculated with non-recombinant MVA, were infected by week two and seroconverted by weeks four to eight. In contrast a sharp increase of both humoral and proliferative responses at two weeks post-challenge was observed in vaccinated monkeys compared to control monkeys. Although all vaccinated monkeys were infected, vaccination with rMVA-J5 appeared to partially control viral replication during the acute and late phase of infection as judged by cell- and plasma-associated viral load.

High virus loads in naturally and experimentally SIVagm-infected African green monkeys.

A quantitative RT-PCR assay was developed for SIVagm and was used to measure the levels of viral RNA in the plasma of experimentally and naturally infected African green monkeys. The number of productively infected PBMCs and the number of cells carrying integrated provirus were also measured. Plasma virus loads in experimentally infected animals peaked at 2 weeks postinfection, ranging from 2.9 x 10(5) to 4.2 x 10(7) RNA copies/ml plasma. Set points of 2.1 x 10(3) to 2.8 x 10(6) RNA copies/ml plasma were maintained for one year. Similarly, the number of cells carrying integrated SIVagm provirus remained relatively stable in individual animals for one year with set points ranging from 73 to 810 proviral copies per 10(6) PBMC. However, the number of productively infected cells fluctuated considerably during this period. Virus loads in the 26 naturally infected AGMs ranged from 8.3 x 10(3) to 1.1 x 10(7) (mean 1.7 x 10(6)) RNA copies/ml plasma. These levels of viremia are similar to those seen in pathogenic systems (HIV-1, SIVmac), indicating that control of SIVagm replication is not the reason for the natural host's resistance to disease progression.

Interleukin-16 stimulates the expression and production of pro-inflammatory cytokines by human monocytes.

Interleukin-16 (IL-16) acts as a chemoattractant for CD4+ cells, as a modulator of T-cell activation, and plays a key role in asthma. This report describes the cytokine-inducing effects of IL-16 on total peripheral blood mononuclear cells (PBMC) and PBMC subpopulations. While CD4+ T lymphocytes did not secrete cytokines in response to rhIL-16, CD14+ CD4+ monocytes and maturing macrophages secrete IL-1beta, IL-6, IL-15 and tumour necrosis factor-alpha (TNF-alpha) upon rhIL-16 stimulation. The mRNA species for these four cytokines were detected as early as 4 hr post-stimulation, with protein being secreted by 24 hr. Secretion of IL-1beta and IL-6 by total PBMC was dose dependent, with maximal secretion being observed using 50 ng/ml rhIL-16. However, for IL-15 or TNF-alpha maximal secretion by total PBMC occurred with all concentrations between 5 ng/ml to 500 ng/ml rhIL-16. Purified monocytes/macrophages secreted maximal concentrations of all four cytokines in the presence of 500 ng/ml rhIL-16, except for monocytes where maximal secretion of IL-15 was, interestingly, observed with only 50 ng/ml rhIL-16. The use of higher concentrations of rhIL-16 (1000 ng/ml) inhibited secretion of all four cytokines. While these IL-16-induced cytokines are likely to be involved in the immune system's response to antigen, the data suggest that IL-16 may play a key role in initiating and/or sustaining an inflammatory response.

Cutting edge: CD4 is not required for the functional activity of IL-16.

IL-16 functions as a chemoattractant factor, inhibitor of HIV replication, and inducer of proinflammatory cytokine production. Previous studies have suggested that CD4 is the receptor for IL-16, because only CD4+ cells respond to IL-16 and both the anti-CD4 Ab OKT4 and soluble CD4 can block IL-16 function. However, these are only indirect evidence of a requirement for CD4, and to date a direct interaction between IL-16 and CD4 has not been shown. In this paper, we report that cells from CD4 knockout mice are as responsive to IL-16 as their CD4 wild-type equivalents in both assays testing for IL-16 function (chemotaxis and production of proinflammatory cytokines). In addition, the inhibitory effect of soluble CD4 on IL-16 function observed using CD4 wild type murine cells was not observed using CD4 knockout cells. These data demonstrate that CD4 is not required for IL-16 function and suggest that another molecule acts as the major receptor.

Two distinct gamma-2 herpesviruses in African green monkeys: a second gamma-2 herpesvirus lineage among old world primates?

Primate gamma-2 herpesviruses (rhadinoviruses) have so far been found in humans (Kaposi's sarcoma-associated herpesvirus [KSHV], also called human herpesvirus 8), macaques (Macaca spp.) (rhesus rhadinovirus [RRV] and retroperitoneal fibromatosis herpesvirus [RFHV]), squirrel monkeys (Saimiri sciureus) (herpesvirus saimiri), and spider monkeys (Ateles spp.) (herpesvirus ateles). Using serological screening and degenerate consensus primer PCR for the viral DNA polymerase gene, we have detected sequences from two distinct gamma-2 herpesviruses, termed Chlorocebus rhadinovirus 1 (ChRV1) and ChRV2, in African green monkeys. ChRV1 is more closely related to KSHV and RFHV, whereas ChRV2 is closest to RRV. Our findings suggest the existence of two distinct rhadinovirus lineages, represented by the KSHV/RFHV/ChRV1 group and the RRV/ChRV2 group, respectively, in at least two Old World monkey species. Antibodies to members of the RRV/ChRV2 lineage may cross-react in an immunofluorescence assay for early and late KSHV antigens.

Decline of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes in the peripheral blood of long-term nonprogressing macaques infected with SIVmac32H-J5.

The evolution of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte precursors (CTLps) and their relationship with virus replication were studied in SIV-infected macaques. After primary viremia, 3 of 8 macaques lost culturable virus and polymerase chain reaction-detectable provirus in peripheral blood. Although proviral DNA persisted in the spleen and lymph nodes, virus loads were below or barely above detection levels. Throughout the study, the 3 macaques remained asymptomatic, with stable CD4+ cell counts. These findings were associated with the detection of CTLps directed against both structural and regulatory SIV proteins. The response peaked during the first 7 months of infection but waned subsequently. CTLps increased after rechallenge of 1 macaque, suggesting that limited antigenic stimulation contributed to their disappearance from circulation. Transient viremia with increasing CTLp frequencies and antibody titers also suggested at least partial susceptibility to reinfection. These findings bear implications for vaccination strategies aimed at inducing protective CTLs against lentiviruses.

Simian immunodeficiency virus (SIV) from sun-tailed monkeys (Cercopithecus solatus): evidence for host-dependent evolution of SIV within the C. lhoesti superspecies.

Recently we reported the characterization of simian immunodeficiency virus (SIVlhoest) from a central African l'hoest monkey (Cercopithecus lhoesti lhoesti) that revealed a distant relationship to SIV isolated from a mandrill (SIVmnd). The present report describes a novel SIV (SIVsun) isolated from a healthy, wild-caught sun-tailed monkey (Cercopithecus lhoesti solatus), another member of the l'hoest superspecies. SIVsun replicated in a variety of human T-cell lines and in peripheral blood mononuclear cells of macaques (Macaca spp.) and patas monkeys (Erythrocebus patas). A full-length infectious clone of SIVsun was derived, and genetic analysis revealed that SIVsun was most closely related to SIVlhoest, with an amino acid identity of 71% in Gag, 73% in Pol, and 67% in Env. This degree of similarity is reminiscent of that observed between SIVagm isolates from vervet, grivet, and tantalus species of African green monkeys. The close relationship between SIVsun and SIVlhoest, despite their geographically distinct habitats, is consistent with evolution from a common ancestor, providing further evidence for the ancient nature of the primate lentivirus family. In addition, this observation leads us to suggest that the SIVmnd lineage should be designated the SIVlhoest lineage.

Why are the natural hosts of SIV resistant to AIDS?

An increasing number of African primate species have been shown to be infected in the wild with their own distinct variants of simian immunodeficiency virus. The most striking feature of these natural host systems is the lack of AIDS-like disease despite long-term infection. In the African green monkey (AGM)/SIVagm system there is no evidence that a vigorous antiviral immune response, a lack of variability or a low virus load accounts for this lack of pathogenicity. New-born AGMs appear to be even more resistant to the virus than adults, despite their immature immune system and higher pool of target cells. The fact that AGMs, unlike HIV-infected humans, lack a humoral immune response to non-denatured Gag protein and do not show trapping of virus in the lymph nodes suggested that tolerance to Gag might prevent the formation of immune complexes which would normally be filtered out by the lymphoid tissues with detrimental results. This apparent tolerance to Gag is a common feature of many, if not all, of the natural host systems and might explain why the lymph nodes and immune system in general remain intact in these primates in the face of continuous, high level virus replication.

Live attenuated simian immunodeficiency virus (SIV)mac in macaques can induce protection against mucosal infection with SIVsm.

OBJECTIVE: To investigate whether vaccination of macaques with attenuated simian immunodeficiency virus (SIV)macC8 could induce long-term protective immunity against rectal exposure to SIVsm and intravenous exposure to the more divergent HIV-2. DESIGN AND METHODS: Eight months after vaccination with live attenuated SIVmacC8, four cynomolgus monkeys were challenged with SIVsm intrarectally and another four vaccinated monkeys were challenged with HIV-2 intravenously. Sixteen months after SIVmacC8 vaccination, another two monkeys were challenged with SIVsm across the rectal mucosa. Two vaccinees shown to be protected against SIVsm were rechallenged 8 months after the first challenge. Ten naive animals were used as controls. Serum antigenaemia, virus isolation, antibody responses, cell-mediated immunity and CD4+ and CD8+ T-cell subpopulations were monitored. PCR-based assays were used to distinguish between virus populations. RESULTS: At the time of challenge, eight out of 10 vaccinees were PCR-positive for SIVmacC8 DNA but no virus could be isolated from peripheral blood mononuclear cells. After SIVsm challenge, three out of six vaccinees were repeatedly SIVsm PCR-negative. In one of the three infected monkeys, the challenge virus was initially suppressed but the monkey ultimately developed AIDS after increased replication of the pathogenic virus. Rechallenged monkeys remained protected. All HIV-2-challenged vaccinees became superinfected. All controls became infected with either SIVsm or HIV-2. At the time of challenge the vaccinees had neutralizing antibodies to SIVmac but no demonstrable cross-neutralizing antibodies to SIVsm or HIV-2. Titres of antigen-binding or neutralizing antibodies did not correlate with protection. Cytotoxic T-cell responses to SIV Gag/Pol and virus-specific T-cell proliferative responses were low. CONCLUSION: The live attenuated SIVmacC8 vaccine was able to induce long-term protection against heterologous intrarectal SIVsm challenge in a proportion of macaques but not against the more divergent HIV-2, which was given intravenously.

Chemoattractant factors and the control of human immunodeficiency virus replication.

Factors secreted by CD8(+) T cells have been described to suppress immunodeficiency virus replication. The research efforts to identify these factors led to the proposal of some candidate proteins as being responsible for the antiviral effects. Chemokines and IL-16 are secreted by CD8(+) T cells and inhibit HIV replication through different mechanisms. However, their antiviral properties cannot fully explain the inhibitory activities found in cell culture supernatants from CD8(+) T cells.

In vivo resistance to simian immunodeficiency virus superinfection depends on attenuated virus dose.

Infection of macaques with attenuated simian immunodeficiency virus (SIV) induces potent superinfection resistance that may be applicable to the development of an AIDS vaccine but little information exists concerning the conditions necessary for the induction of this vaccine effect. We report that only a high dose of attenuated SIVmac protected macaques against intravenous challenge with more virulent virus 15 weeks after primary infection. Three of four animals given 2000-20000 TCID50 of SIVmacC8, a molecular clone of SIVmac251(32H) with a 12 bp deletion in the nef gene, essentially resisted superinfection with uncloned SIVmac. In two animals challenge virus was never detected by PCR and in one animal challenge virus was detected on one occasion only. Although animals given 2-200 TCID50 of attenuated virus were superinfected they were spared from the loss of CD4 cells seen in infected naive controls. Protection from superinfection did not correlate with immune responses, including the levels of virus-specific antibodies or virus-neutralizing activity measured on the day of challenge; although, after superinfection challenge, Nef-specific CTL responses were detected only in animals infected with high doses of attenuated SIV. Unexpectedly, cell-associated virus loads 2 weeks after inoculation were significantly lower in animals infected with a high dose of attenuated SIV compared to those in animals infected with a low dose. Our results suggest that the early dynamics of infection with attenuated virus influence superinfection resistance.

Simian immunodeficiency virus of African green monkeys is apathogenic in the newborn natural host.

Several studies have demonstrated that newborn animals are more susceptible to disease development following infection with retroviruses than adults. Adult African green monkeys (AGMs) infected with SIVagm do not develop AIDS-like disease and the objective of the study was to determine whether experimental infection of newborn AGMs with SIVagm would result in pathogenesis. Neonatal AGMs were found to have a higher percentage of circulating CD4+ lymphocytes than adults (62% versus 14%) and therefore a higher potential pool of target cells for SIVagm infection. However, no differences in the in vitro replication kinetics of SIVagm in peripheral blood mononuclear cells of adult or neonatal AGMs could be observed. In vivo, the neonatal AGMs became viremic at the earliest two months after inoculation whereas the adult AGMs had evidence of virus replication already 2 to 6 weeks after infection. None of the animals developed AIDS-like symptoms upon infection. In the heterologous cynomolgus macaque host, a newborn infected with SIVagm developed early high virus loads and died two months after birth with AIDS-like histopathologic features. It would therefore appear that in contrast to the situation with many other retroviruses, newborn AGMs are no more permissive to SIVagm infection than are adults.

The U3 promoter and the nef gene of simian immunodeficiency virus (SIV) smmPBj1.9 do not confer acute pathogenicity upon SIVagm.

Two chimeric proviruses comprising the U3 promoter and the nef gene of simian immunodeficiency virus (SIV) smmPBj1.9 in addition to other genomic regions of SIVagm3mc from African green monkeys (Cercopithecus aethiops) were constructed. The derived chimeric viruses (SIVagm3mc/SIVsmmPBj1.9) were both able to replicate in nonstimulated peripheral blood leukocytes from pig-tailed macaques (Macaca nemestrina), a biological property often correlated with acute pathogenicity. However, only one of the chimeric viruses was acutely pathogenic, inducing a rapid depletion of the peripheral CD4+ T cells in two infected pig-tailed macaques within 10 days after infection in a manner similar to infection with SIVsmmPBj1.9 itself. The other chimeric virus actively replicated during the first 8 weeks after experimental infection of two pig-tailed macaques but induced neither acute disease nor CD4+ T-cell depletion for 113 weeks after infection. Thus, the U3 promoter and the nef gene of SIVsmmPBj1.9 alone appear to be insufficient to confer acute pathogenicity to SIVagm3mc.

SIVmac vaccine studies using whole inactivated virus antigen sequentially depleted of viral proteins.

Groups of four rhesus monkeys were immunised at 0, 1, 2, and 13 months with whole inactivated SIVmac32H, SIVmac depleted of the outer envelope glycoprotein gp130, virus cores depleted of the lipid membrane (and hence transmembrane glycoproteins), or purified gag protein. These macaques plus controls were challenged with either the homologous SIVmac251-32H grown in human cells or the same virus passed once through monkey cells. None of those challenged with monkey-grown virus were protected, whereas all in the whole and gp130-depleted virus groups, and one in the core group resisted challenge with human-grown virus. As the only difference between the challenge viruses was a single in vitro passage in monkey cells it can be concluded that protection was solely due to human cell components. Finally, passive transfer of high titer IgG from monkeys infected with the homologous challenge virus failed to protect monkeys from infection despite the presence of circulating neutralising antibodies.