Genital-associated lymphoid tissue in female non-human primates.

We investigated genital-associated lymphoid tissue (GENALT) in non-human primates (macaques), by augmenting vaginal with oral immunization. The vaccine was a recombinant particulate SIV antigen (p27:Ty-VLP), linked to CT-B, and administered into the vagina by a paediatric naso-gastric tube and into the stomach by a gastric tube. Oro-vaginal or vagino-oral sequence of immunization elicited specific CD4+ T cell proliferative responses to p27 antigen in the genital lymph nodes and the spleen but not in unrelated lymph nodes. CD4+ T cells reconstituted with B cells and macrophages from the genital lymph nodes induced specific IgA and to a lesser extent IgG anti-p27 antibodies. However, the corresponding splenic cells induced greater IgG than IgA antibody synthesis. Intramuscular immunization primed splenic but not genital lymph node cells, and induced CD4+ T cell proliferative responses and predominantly B cell IgG antibody synthesis. Finding primed B and T cells in the genital lymph nodes after augmenting vaginal by oral immunization provides experimental evidence for GENALT in non-human primates. This primate model of vaginal immunization suggests 3 levels of specific immunity: (1) secretory IgA (and IgG) in the cervico-vaginal mucosal epithelium; (2) primed CD4+ T cells and B cells in the genital lymph nodes and the spleen; and (3) circulating CD4+ T cells, B cells and IgG and IgA antibodies specific to the immunizing antigen.

T cell responses in macaques after vaginal immunization with particulate SIV p27 antigen.

Rhesus monkeys were immunized by the vaginal and oral routes using a recombinant particulate simian immunodeficiency virus (SIV) antigen. Augmenting vaginal by oral immunization in macaques elicits proliferative CD4+ T cells in the circulation which are specific to the immunizing p27 antigen. Reconstitution of enriched CD4+ T cells, B cells and macrophages from circulating mononuclear cells help B cells in specific IgA anti-p27 antibody synthesis. The results suggest that augmented vaginal immunization induces systemic CD4+ T and B cell responses which may play a part in the protective immunity against SIV (HIV) infection.

Targeted lymph node immunization with simian immunodeficiency virus p27 antigen to elicit genital, rectal, and urinary immune responses in nonhuman primates.

A s.c. route of immunization was developed in non-human primates, which targets the genitourinary-rectal associated lymphoid tissue. A vaccine consisting of rSIV gag p27, expressed as hybrid Ty virus-like particles (p27: Ty-VLP) was administered in the proximity of the internal iliac lymph nodes. Secretory IgA and IgG Abs to the p27 Ag were elicited in the vaginal, male urethral, rectal and seminal fluids, urine and serum. Two or more immunodominant B cell epitopes were identified within peptides 51-90 and 121-170 of the sequence of p27, using serum or biliary IgA and IgG Abs. CD4+ T cell proliferative responses to p27 were elicited predominantly in the targeted internal iliac, as well as the inferior mesenteric lymph nodes and the spleen, but not in the unrelated lymph nodes. These cells were then studied for helper function in p27 specific B cell Ab synthesis. Specific IgA and IgG Abs were detected in the same lymphoid tissues as those that displayed proliferative responses. However, cross-over reconstitution experiments between splenic and iliac lymph node B and CD4+ T cells suggest that the iliac B cells are essential for specific IgA Ab synthesis, whereas splenic B cells preferentially synthesize IgG Ab. The targeted lymph node (TLN) route of immunization gave comparable B cell, proliferative T cell, and Th cell responses to the vaginal, male genitourinary, and rectal mucosal routes, which were augmented by oral immunization. However, the TLN route induced urinary and seminal fluid sIgA and IgG Abs in addition to genital and rectal Abs. Generating secretory IgA and IgG Abs at the mucosal surfaces, and T and B cell immunity in the regional draining lymph nodes, spleen and circulation by TLN immunization may prevent transmission of virus through the mucosa, dissemination of the virus, and the formation of a latent reservoir of infection.

Mucosal model of genital immunization in male rhesus macaques with a recombinant simian immunodeficiency virus p27 antigen.

Human immunodeficiency virus (HIV) can be transmitted through infected seminal fluid or vaginal or rectal secretions during heterosexual or homosexual intercourse. To prevent mucosal transmission and spread to the regional lymph nodes, an effective vaccine may need to stimulate immune responses at the genitourinary mucosa. In this study, we have developed a mucosal model of genital immunization in male rhesus macaques, by topical urethral immunization with recombinant simian immunodeficiency virus p27gag, expressed as a hybrid Ty virus-like particle (Ty-VLP) and covalently linked to cholera toxin B subunit. This treatment was augmented by oral immunization with the same vaccine but with added killed cholera vibrios. Polymeric secretory immunoglobulin A (sIgA) and IgG antibodies to p27 were induced in urethral secretions, urine, and seminal fluid. This raises the possibility that the antibodies may function as a primary mucosal defense barrier against SIV (HIV) infection. The regional lymph nodes which constitute the genital-associated lymphoid tissue contained p27-specific CD4+ proliferative and helper T cells for antibody synthesis by B cells, which may function as a secondary immune barrier to infection. Blood and splenic lymphocytes also showed p27-sensitized CD4+ T cells and B cells in addition to serum IgG and IgA p27-specific antibodies; this constitutes a third level of immunity against dissemination of the virus. A comparison of genito-oral with recto-oral and intramuscular routes of immunization suggests that only genito-oral immunization elicits specific sIgA and IgG antibodies in the urine, urethra, and seminal fluid. Both genito-oral and recto-oral immunizations induced T-cell and B-cell immune responses in regional lymph nodes, with preferential IgA antibody synthesis. The mucosal route of immunization may prevent not only virus transmission through the genital mucosa but also dissemination and latency of the virus in the draining lymph nodes.

T- and B-cell functions and epitope expression in nonhuman primates immunized with simian immunodeficiency virus antigen by the rectal route.

Transmission of human immunodeficiency virus (HIV) in North America and Europe occurs most commonly through the rectal mucosa during homosexual intercourse. The simian immunodeficiency virus (SIV) macaque model has been used to investigate rectal immunization. The vaccine used was a recombinant SIV gag p27 expressed as hybrid Ty virus-like particles (Ty-VLP). Sequential ororectal (OR) mucosal immunization was compared with i.m. immunization. Whereas both routes of immunization induced serum IgA and IgG p27 antibodies, only OR immunization induced rectal secretory IgA antibodies. Specific CD4+ T-cell proliferative responses to stimulation with p27 were found after i.m. immunization only in the blood and spleen, but after OR immunization they were found in the internal iliac and inferior mesenteric lymph nodes in addition to the blood and spleen. T-cell epitope mapping of the proliferative responses of short-term cell lines (STCLs) grown from peripheral blood or lymphoid cells revealed a major epitope within the polypeptide 121-150 after either route of immunization. Two minor T-cell epitopes were found within peptide 41-80 in STCLs from splenic and circulating cells. B-cell epitope mapping of serum or biliary IgA and IgG antibodies revealed two overlapping or adjacent immunodominant epitopes to the T-cell epitopes within the polypeptides 121-170 and 51-90. The results suggest that rectal augmented by oral immunization with a recombinant particulate antigen in nonhuman primates elicits secretory IgA and to a lesser extent IgG responses in the draining lymph nodes and the rectal mucosa, whereas systemic immunization targets predominantly splenic and circulating T- and B-cell responses. These findings may have important implications in the strategy of designing vaccines in prevention of homosexual transmission of HIV infection.

Induction of mucosal and systemic immunity to a recombinant simian immunodeficiency viral protein.

Heterosexual transmission through the cervico-vaginal mucosa is the principal route of human immunodeficiency virus (HIV) infection in Africa and is increasing in the United States and Europe. Vaginal immunization with simian immunodeficiency virus (SIV) had not yet been studied in nonhuman primates. Immune responses in macaques were investigated by stimulation of the genital and gut-associated lymphoid tissue with a recombinant, particulate SIV antigen. Vaginal, followed by oral, administration of the vaccine elicited three types of immunity: (i) gag protein p27-specific, secretory immunoglobulin A (IgA) and immunoglobulin G (IgG) in the vaginal fluid, (ii) specific CD4+ T cell proliferation and helper function in B cell p27-specific IgA synthesis in the genital lymph nodes, and (iii) specific serum IgA and IgG, with CD4+ T cell proliferative and helper functions in the circulating blood.

Relation between humoral responses to HIV gag and env proteins at seroconversion and clinical outcome of HIV infection.

OBJECTIVE: To study the contribution of the humoral response to HIV-I at seroconversion to disease outcome after 84 months. DESIGN: A retrospective longitudinal study. SETTING: Two haemophilia centres in the United Kingdom. PATIENTS: 88 Haemophiliac patients infected with HIV-I for whom sera were available from before seroconversion and in whom clinical follow up data were available. RESULTS: Kaplan-Meier survival analysis showed a significant difference between a high titre (greater than 1600) p24 antibody response at seroconversion and prolonged time before the development of HIV related disease (p = 0.0008). In contrast, higher titres of antibody to gp120 at seroconversion (greater than 25,600) correlated with more rapid clinical deterioration (p = 0.025). CONCLUSIONS: The first humoral response to HIV proteins at seroconversion is associated with clinical outcome; patients with an initial low titre antibody response to the gagp24 protein have a significantly faster rate of progression to CDC stage IV disease. Patients with a high titre p24 antibody response progress to AIDS more slowly, and these data provide an explanation why p24 antigenaemia is not universally detected in patients with AIDS. It is unclear whether the association between a strong initial p24 antibody response and slower progression of HIV disease is causal and if so whether it is due to viral or host factors.

Antibodies to HIV-1 nef(p27): prevalence, significance, and relationship to seroconversion.

A sensitive and specific enzyme-linked immunoassay for antibodies to the human immunodeficiency virus type 1 (HIV-1) nef gene product, p27, has been developed using recombinant Escherichia coli-derived protein from the LAV-1-Bru sequence. Of 92 HIV-1 infected hemophiliacs, 72 (78%) produced anti-nef antibodies in this assay; the early appearance of anti-nef prior to full seroconversion was a rare event in this population, occurring in only one subject (approximately 1%). Anti-nef antibodies were not detected in any of 500 sera from 98 repeatedly HIV seronegative subjects who had been exposed to sexually transmitted modes of HIV infection (45 subjects) or through blood products (53 subjects). There was no significant association of titer or anti-nef antibody with protection from disease in HIV infection (p = 0.1). Although the nef protein is relatively immunogenic in natural infection, this study cannot confirm the previously reported high prevalence of anti-nef antibodies prior to seroconversion, nor the finding of anti-nef antibodies in HIV seronegative but exposed subjects.

Antibodies to a putative HIV gp41 immunosuppressive peptide, pHIVIS (583-599), do not correlate significantly with outcome in HIV infection.

We have studied the prevalence of antibodies to peptides derived from the transmembrane protein of HIV, gp41. Previous work has suggested that the presence of antibodies to the gp41 peptide known as pHIVIS (env 583-599) is associated with protection from immunosuppression in HIV infection. We studied 171 sequential sera from 55 HIV-1-infected people in various clinical stages of disease. There was no significant association between antibodies to pHIVIS and clinical status in this study. Although pHIVIS has sequence similarity to the putative immunosuppressive region of the C-type oncornaviruses (p15E), antibodies to this peptide do not appear to be associated with protection from immunosuppression in natural HIV infection.

Immunoadjuvant action of liposomes: comparison with other adjuvants.

Dehydration-rehydration vesicles (DRV liposomes) composed of equimolar phospholipid and cholesterol and containing bovine serum albumin (BSA) were used together with free BSA to immunize Balb/C mice. Primary and secondary immune responses (IgG1) to the liposomal antigen, as measured by ELISA in mouse sera, were similar for egg phosphatidylcholine (PC) and distearoyl phosphatidylcholine (DSPC) DRV, and much greater than those elicited by free BSA. The adjuvanticity of PC DRV was compared with that of aluminium salts (alum), complete Freund's adjuvant (CFA) and N-acetyl muramyl-L-threonyl-D-isoglutamine ([Thr1]MDP), the latter used as such or in a liposome form co-entrapped with the antigen. DRV (with or without co-entrapped [Thr1]MDP), and alum were equally strong in producing primary and secondary immune responses (IgG1) to BSA. Such responses were significantly higher than those achieved with CFA and [Thr1]MDP alone. The implications of these results for the potential role of liposomes as immunological adjuvants in vaccines are discussed.