Comparison of systemic and mucosal vaccination: impact on intravenous and rectal SIV challenge.

Mucosal tissues are the primary route of transmission for most respiratory and sexually transmitted diseases, including human immunodeficiency virus. We aimed to generate strong mucosal immune responses to simian immunodeficiency virus (SIV) in rhesus macaques by targeting recombinant adenovirus serotype 5 (rAd5) to the lung. The immunogenicity and efficacy of aerosol (AE) vaccination was compared with intramuscular (IM) delivery in either an intravenous (IV) or intrarectal (IR) SIV(mac251) challenge model. Aerosolized rAd5 induced strong cellular responses in the lung and systemic humoral responses equivalent to IM. Strikingly, all immunization groups controlled acute viremia in the IV challenge model by 1-2 logs. By contrast, after IR challenge, only peak viremia was reduced by immunization, with no significant effect on SIV infection acquisition rate or mucosal CD4(+) T-cell preservation. Improved disease outcome was associated with pre-challenge cellular and humoral responses, while post-challenge T-cell responses were highly correlated with viremia control. The similar outcomes achieved by systemic and airway mucosal immunization support AE delivery as a safe, effective, and less invasive alternative to parenteral vaccination.

Nasal DNA-MVA SIV vaccination provides more significant protection from progression to AIDS than a similar intramuscular vaccination.

Preventive human immunodeficiency virus (HIV) vaccination may require induction of virus-specific immune responses at mucosal sites to contain viral infection locally after exposure, as most HIV infections occur through mucosal surfaces. We compared the efficacy of an intranasal or intramuscular Simian immunodeficiency virus (SIV)+ interleukin (IL)-2+IL-15 DNA/SIV-MVA (modified vaccinia virus Ankara) vaccination in preventing disease progression in SIVmac251 intrarectally challenged rhesus macaques. SIV-specific rectal IgA responses were more significantly persistent in nasally vaccinated than in intramuscularly vaccinated animals. No significant differences were observed in the magnitude of systemic T-cell responses between the two groups, although the nasal immunization induced more significant anti-SIV T-cell responses in the colorectal mucosa. After challenge, CD4(+) central memory (C(M)) T-cell preservation and significant disease-delay were observed in both vaccination groups. However, nasally vaccinated animals had more significant early preservation of circulating and colorectal CD4(+) C(M) T cells, of circulating CD4(+)/alpha4beta7(+) effector memory (E(M)) T cells, and a longer disease-free interval when compared with the intramuscularly vaccinated or control groups. Regardless of vaccination status, long-term viremia control and preservation of CD4(+) C(M) T cells was detected in animals with significantly higher systemic CD8(+)/tumor necrosis factor (TNF)-alpha(+) and CD8(+)/interferon (IFN)-gamma(+) T-cell responses and higher SIV-specific CD4(+)/IL-2(+) responses in colorectal T cells.

Immunization of mice with recombinant gp41 in a systemic prime/mucosal boost protocol induces HIV-1-specific serum IgG and secretory IgA antibodies.

We tested the immunogenicity in mice of a recombinant fusion protein (gp41HA) consisting of the ectodomain of the HIV-1(IIIB) envelope glycoprotein gp41 fused to a fragment of the influenza virus HA2 hemagglutinin protein. An intraperitoneal prime followed by intranasal or intragastric boosts with gp41HA induced high concentrations of serum IgG antibodies and fecal IgA antibodies that reacted with gp41 in HIV-1(IIIB) viral lysate and were cross-reactive with gp41 in HIV-1(MN) lysate. By indirect immunofluorescence, serum IgG and fecal IgA from immunized mice were also shown to recognize gp41 in acetone-fixed human peripheral blood mononuclear cells infected with either syncytium-inducing (SI) or non-syncytium-inducing (NSI) North American HIV-1 field isolates, but not uninfected cells. Thus, this recombinant antigen may be useful in prime/boost immunization protocols designed to induce systemic and mucosal antibodies that recognize multiple primary HIV-1 isolates.

Modified wick method using Weck-Cel sponges for collection of human rectal secretions and analysis of mucosal HIV antibody.

Weck-Cel sponges were examined for suitability as an absorbent material for nontraumatic collection of rectal secretions in humans. Sponges were tested in vitro and determined by quantitative enzyme-linked immunosorbent assay (ELISA) to be capable of releasing 100% of absorbed albumin and all immunoglobulin subtypes after treatment with detergent-supplemented buffer. Protein composition in rectal secretions collected from normal women with dry sponges (DS) or with sponges previously softened by moistening with saline (MS) was subsequently compared. DS secretions showed evidence of contamination with blood and interstitial fluid-derived albumin, immunoglobulin G (IgG), and monomeric IgA. MS secretions appeared to represent local mucosal secretions more accurately because they contained negligible blood, a greater percentage of secretory IgA within the total IgA, and both lower albumin/IgG ratios and more dramatic alterations in IgG subclass distribution compared with corresponding serum. Anti-HIV IgG, IgM, IgA, and antibodies with secretory component could be demonstrated by ELISA in rectal secretions collected with moist sponges from 8 of 8, 1 of 8, 5 of 8, and 3 of 8 HIV-infected women, respectively. The data show that Weck-Cel sponges, if premoistened, can be used to collect rectal fluids nontraumatically and to obtain quantitative information about concentrations of immunoglobulins and specific antibodies on rectal mucosal surfaces.

Effective induction of simian immunodeficiency virus-specific systemic and mucosal immune responses in primates by vaccination with proviral DNA producing intact but noninfectious virions.

We report a pilot evaluation of a DNA vaccine producing genetically inactivated simian immunodeficiency virus (SIV) particles in primates, with a focus on eliciting mucosal immunity. Our results demonstrate that DNA vaccines can be used to stimulate strong virus-specific mucosal immune responses in primates. The levels of immunoglobulin A (IgA) detected in rectal secretions of macaques that received the DNA vaccine intradermally and at the rectal mucosa were the most striking of all measured immune responses and were higher than usually achieved through natural infection. However, cytotoxic T lymphocyte responses were generally low and sporadically present in different animals. Upon rectal challenge with cloned SIVmac239, resistance to infection was observed, but some animals with high SIV-specific IgA levels in rectal secretions became infected. Our results suggest that high levels of IgA alone are not sufficient to prevent the establishment of chronic infection, although mucosal IgA responses may have a role in reducing the infectivity of the initial viral inoculum.

Immunization of mice with peptomers covalently coupled to aluminum oxide nanoparticles.

Subunit vaccines generally require adjuvants to elicit immune responses, but adjuvants may alter the conformation of critical epitopes and reduce vaccine efficacy. We therefore tested an immunization strategy in which antigen is covalently coupled to aluminum oxide nanoparticles using a method that favors preservation of the native conformation. The test antigen consisted of "peptomers" (head-to-tail-linked peptide homopolymers) derived from the 4th conserved region (C4) of HIV-1 gp120 which is believed to be in an alpha-helical conformation prior to binding to CD4. Immune responses in mice to peptomer-nanoparticle conjugates were compared to responses elicited by free C4 peptide and C4 peptomers, with and without the hydrophilic adjuvant muramyl dipeptide (MDP). Highest peptomer-specific serum antibody responses were induced by peptomer-particles without MDP. Serum antibodies induced by peptomer-particles also showed highest reactivity towards recombinant, glycosylated gp120 and HIV-1 infected T cells. The results suggest that this novel vaccine approach could be useful for induction of immune responses against conformation-sensitive viral antigens without the need for additional adjuvants.

Mucosal vaccination strategies for women.

Women were immunized orally, rectally, or vaginally with a recombinant cholera toxin B-containing vaccine to determine which of these mucosal immunization routes generate the greatest levels of antibody in the female genital tract and rectum. ELISA was used to measure concentrations of cholera toxin B-specific IgA and IgG antibody in serum and secretions before and after three immunizations. Each immunization route similarly increased specific IgG in serum and specific IgA in saliva. Only the vaginal route increased IgA antibodies in genital tract secretions and could be shown to induce a local IgG response. However, vaginal immunization failed to produce antibody in the rectum. In a similar fashion, rectal immunization elicited highest concentrations of locally derived IgA and IgG antibody in the rectum but was ineffective for generating antibody in the genital tract. The data suggest that local immunization may induce the greatest immune responses in the female genital tract and rectum of humans.

Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women.

To determine which mucosal immunization routes may be optimal for induction of antibodies in the rectum and female genital tract, groups of women were immunized a total of three times either orally, rectally, or vaginally with a cholera vaccine containing killed Vibrio cholerae cells and the recombinant cholera toxin B (CTB) subunit. Systemic and mucosal antibody responses were assessed at 2-week intervals by quantitation of CTB-specific antibodies in serum and in secretions collected directly from mucosal surfaces of the oral cavity, rectum, cervix, and vagina with absorbent wicks. The three immunization routes increased levels of specific immunoglobulin G (IgG) in serum and specific IgA in saliva to similar extents. Rectal immunization was superior to other routes for inducing high levels of specific IgA and IgG in rectal secretions but was least effective for generating antibodies in female genital tract secretions. Only vaginal immunization significantly increased both specific IgA and specific IgG in both the cervix and the vagina. In addition, local production of CTB-specific IgG in the genital tract could be demonstrated only in vaginally immunized women. Vaginal immunization did not generate antibodies in the rectum, however. Thus, generation of optimal immune responses to sexually transmitted organisms in both the rectal and the genital mucosae of women may require local immunization at both of these sites.

High prevalence of serum IgA HIV-1 infection-enhancing antibodies in HIV-infected persons. Masking by IgG.

IgA and IgG purified from sera of 20 HIV-infected persons were separately examined for ability to mediate Ab-dependent enhancement (ADE) of HIV-1 infection of U937 cells. Both isotypes were capable of enhancing infection of these cells. However, IgA from twice as many persons (14/20) displayed infection enhancement when compared with IgG. This activity was predominantly observed with IgA from asymptomatic HIV-seropositive subjects (9/9). Enhanced HIV infection by IgA was observed most often at concentrations equivalent to serum dilutions in the range 10(-3) to 10(-5) and could be inhibited by preincubation of U937 cells with a mAb specific for the Fc alpha receptor. Concentrations of IgG mediating ADE were generally present in sera at dilutions from 10(-4) to 10(-6). When IgG was adjusted to physiologic concentration and combined with an enhancing concentration of IgA, enhancement was not observed unless IgG was also present at a concentration which exhibited this activity. These results suggest that, in comparison with IgG, HIV-infected individuals more often produce IgA Abs reacting with enhancing determinants of HIV. IgA-mediated ADE of HIV infection may not play a significant role in facilitating systemic dissemination of HIV because of the presence of higher concentrations of IgG. However, the production of IgA HIV-enhancing Abs at mucosal sites, where fewer IgG plasma cells are present, could contribute to the pathogenesis of HIV infection and interfere with development of vaccines designed to induce HIV IgA Abs at mucosal surfaces.

A polarized human endometrial cell line that binds and transports polymeric IgA.

We have demonstrated that a human endometrial cell line, HEC-1, maintains a transepithelial electrical resistance, directionally transports fluids across the cell monolayer, and releases enveloped viruses at distinct plasma membrane domains: influenza virus is released at the apical surfaces and vesicular stomatitis virus (VSV) at the basolateral surfaces. In addition, we have examined the expression of domain-specific endogenous proteins, including the polyimmunoglobulin receptor. Multiple endogenous polypeptides were found to be secreted into the culture medium at basolateral surfaces, whereas no secretion of specific polypeptides was observed from apical cell surfaces. Distinct patterns of endogenous proteins were also observed on apical and basolateral cell surfaces, with a much more complex polypeptide pattern on the basolateral membranes. Using surface biotinylation and immunofluorescence, the polyimmunoglobulin receptor was found to be expressed on the basolateral surface of HEC-1 monolayers. The specific binding of poly-immunoglobulin A (pIgA) was found to occur on the basolateral surface, and was followed by transcytosis to the apical surface and release into the apical medium. The observed characteristics indicate that the endometrium-derived HEC-1 epithelial cell line can be employed as a model for studies of protein transport in polarized epithelial cells of human endometrial tissues, as well as for studies of the interaction of microorganisms with epithelial cells in the genital tract.

Contrasting IgA and IgG neutralization capacities and responses to HIV type 1 gp120 V3 loop in HIV-infected individuals.

Quantitative analysis for HIV-1-specific antibodies present in IgA and IgG preparations purified from the serum of HIV-seropositive individuals indicated that the proportion of HIV-specific antibodies present within the IgG isotype was seven times greater than the proportion of IgA HIV antibodies present within the IgA isotype. Dilution of IgA HIV-specific antibodies by nonspecific IgA was observed in patients with elevated serum IgA concentrations, whereas proportions of IgG HIV antibodies rose with increases in concentrations of serum IgG. Although proportions of IgA HIV antibodies were not observed to correlate with the CD4 counts of the individuals from whom immunoglobulins were purified, a significant association between the numbers of such cells and proportion of HIV antibodies present in the IgG isotype was found. Equivalent amounts of IgG were also more effective than IgA at inhibiting HIV-1IIIB infection of a susceptible T cell line. This may be due to the presence of higher proportions of IgG antibodies directed toward non-V3 determinants because reactivity against an HIV-1IIIB V3 peptide was low and did not differ significantly between these isotopes. IgA antibodies reacting against a V3 peptide containing the HIV consensus sequence could be detected in the majority of IgA samples purified from infected individuals. Proportions of IgG consensus V3-specific antibodies within the purified IgG samples were, however, much higher. The presence of accompanying increases in serum IgG concentration and proportions of IgG HIV antibodies, higher proportions of both HIV- and consensus V3-specific antibodies within this isotype, and more effective neutralization by IgG suggests that an HIV-driven response is dominated by B cells committed to production of this immunoglobulin isotype. The observed low proportions of HIV antigen-specific IgA antibodies with dilution in many individuals by elevations in non-HIV-specific IgA suggests that IgA B cells may be more susceptible to factors that mediate the polyclonal activation believed to be responsible for many of the B cell disorders characteristic of HIV infection.

Serum IgA subclasses and molecular forms in HIV infection: selective increases in monomer and apparent restriction of the antibody response to IgA1 antibodies mainly directed at env glycoproteins.

In a study population representing different CDC stages of HIV infection, 58% exhibited IgA hypergammaglobulinemia resulting from proportional increases in both the IgA1 and the IgA2 subclasses. These increases were detected early in infection, did not correlate with CD4 count, and remained elevated throughout disease progression. Absolute concentrations of polymeric IgA present within each subclass were unchanged, indicating that increased production of monomeric IgA1 and IgA2 were responsible for elevations of total IgA. These elevations were not completely attributable to a specific antibody response to viral infection, since Western blot analysis of purified IgA samples indicated that HIV-reactive IgA antibodies could be demonstrated only within the IgA1 subclass. Dominating IgA1 anti-HIV responses were also observed in two secretory IgA samples isolated from colostrum of healthy HIV seropositive mothers, suggesting that a similar isotype restriction exists in the mucosal IgA compartment. The binding of IgA1 to HIV proteins contrasted markedly to that observed with identical concentrations of IgG purified from the sera of the same patients. While IgG reacted more intensely and broadly with all HIV proteins, IgA1 antibodies were directed predominantly against envelope glycoproteins. In many patients, a total lack of IgA1 reactivity to gag and pol proteins was accompanied by intact IgG responses to these same antigens. Though all IgA samples examined reacted with HIV, fewer responses to gp160, gp120, and p24 were observed in samples from AIDS and AIDS-related complex (ARC) patients, suggesting a declining titer of IgA antibodies against these antigens may be associated with disease progression.(ABSTRACT TRUNCATED AT 250 WORDS)