Durdana Rahman

Biodegradable microparticles for oral immunization

Ovalbumin (OVA) was entrapped in poly(lactide-co-glycolide) microparticles and administered to mice. Following intraperitoneal immunization, the microparticles induced both proliferative T-cell responses and cytotoxic T-cell responses in spleen cells. Following oral immunization, the mean salivary IgA antibody response to microparticles was significantly greater than the response to soluble OVA (p < 0.0001). Serum IgG antibody levels were also significantly greater in the group administered microparticles (p < 0.001). Cholera toxin B subunit was also entrapped in microparticles. Following oral immunization in mice, specific antibody-secreting cells were detected both in the spleens and in the mesenteric lymph nodes.

Salivary, gut, vaginal and nasal antibody responses after oral immunization with biodegradable microparticles.

The aims of this study were to determine whether oral immunization with microparticles might lead to a common mucosal response including vaginal secretions. Female Balb/c mice were immunized orally with microparticles containing ovalbumin at 0 and 4 weeks or with soluble antigen. Antibody responses were assayed by ELISA in saliva, gut washings, vaginal washings and serum, and antibody producing cells were assayed by ELISPOT in salivary glands and nasal cavity. After primary immunization, IgA antibodies were detected in vaginal washings, saliva and in gut washings which were significantly greater than those detected with soluble antigen (P < 0.01). After secondary immunization, greatly enhanced antibody titres were found in three fluids. The specific activity (antibody per microgram IgA) of antibodies in vaginal fluid and saliva was significantly greater than in serum or gut wash (P < 0.01). Oral immunization also resulted in the development of antibody forming cells in salivary glands and in nasal associated mucosal tissue. Immunization with microparticles containing antigen should prove useful in immunization against infections affecting a number of different mucosal surfaces.

Transmission of IgA and IgG monoclonal antibodies to mucosal fluids following intranasal or parenteral delivery

Background: The efficacy by which passive antibodies can reach the lungs could be important for the outcome of immunotherapy of respiratory pulmonary infections. We examined how transmission to a number of mucosal sites is affected by the route of inoculation. Methods: Transmission of newly raised IgA class Mabs against mycobacterial surface antigens to saliva, lung or vaginal lavage, bile and serum of BALB/c mice was compared with existing IgG Mabs. ELISA was used for testing body fluids obtained 1–24 h after intranasal or intravenous inoculation and 1–7 days following back-pack tumour growth of hybridomas. Results: Intranasal inoculation resulted in a rapid rise and high levels of both IgA and IgG class Mabs in lung lavage. In contrast, following intravenous Mab injection or back-pack tumour growth of hybridoma cells, effective lung transmission was observed for the IgG1 and IgG2b MAbs, but not for the IgA Mabs. The secretory component was acquired by the transmitted IgA MAbs in the mucosal fluids, but not in the serum. Nevertheless, the time course of mucosal IgA antibody levels was similar to that of the tested IgG Mabs. Furthermore, the relative proportion of transmission to saliva and bile varied between individual Mabs indicating a role of tissue-specific, immunoglobulin class-unrelated mechanisms. Conclusions: Intranasal, rather than parenteral inoculation of mice is required for the efficient delivery of IgA antibodies against respiratory pulmonary pathogens. Interestingly, IgA-secretory component complexing of intranasally applied Mabs did not significantly influence their persistence in the lungs.

Serum and salivary IgA antibody responses to Saccharomyces cerevisiae, Candida albicans and Streptococcus mutans in orofacial granulomatosis and Crohn's disease

Orofacial granulomatosis (OFG) is a condition of unknown aetiology with histological and, in some cases, clinical association with Crohns disease (CD). However, the exact relationship between OFG and CD remains uncertain. The aim of this study was to determine whether OFG could be distinguished immunologically from CD by comparing non‐specific and specific aspects of humoral immunity in serum, whole saliva and parotid saliva in three groups of patients: (a) OFG only (n = 14), (b) those with both oral and gut CD (OFG + CD) (n = 12) and (c) CD without oral involvement (n = 22) and in healthy controls (n = 29). Non‐specific immunoglobulin (IgA, SigA, IgA subclasses and IgG) levels and antibodies to whole cells of Saccharomyces cerevisiae, Candida albicans and Streptococcus mutans were assayed by enzyme‐linked immunosorbent assay (ELISA) in serum, whole saliva and parotid saliva. Serum IgA and IgA1 and IgA2 subclasses were raised in all patient groups (P < 0·01). Salivary IgA (and IgG) levels were raised in OFG and OFG + CD (P < 0·01) but not in the CD group. Parotid IgA was also raised in OFG and OFG + CD but not in CD. The findings suggest that serum IgA changes reflect mucosal inflammation anywhere in the GI tract but that salivary IgA changes reflect involvement of the oral cavity. Furthermore, the elevated levels of IgA in parotid saliva suggest involvement of the salivary glands in OFG. Serum IgA antibodies to S. cerevisiae were raised markedly in the two groups with gut disease while serum IgA (or IgG) antibodies to C. albicans were elevated significantly in all three patient groups (P < 0·02). No differences were found with antibodies to S. mutans. Whole saliva IgA antibodies to S. cerevisiae (and C. albicans) were raised in the groups with oral involvement. These findings suggest that raised serum IgA antibodies to S. cerevisiae may reflect gut inflammation while raised SIgA antibodies to S. cerevisiae or raised IgA or IgA2 levels in saliva reflect oral but not gut disease. Analysis of salivary IgA and IgA antibodies to S. cerevisiae as well as serum antibodies in patients presenting with OFG may allow prediction of gut involvement.

Immunogenic and tolerogenic signatures in human immunodeficiency virus (HIV)-infected controllers compared with progressors and a conversion strategy of virus control.

Epidemiological studies have identified a small cohort of controllers of human immunodeficiency virus (HIV)‐1 infection, who without treatment have no detectable virus, and others who progress at a variable rate. The objective of this study was to distinguish immune signatures in HIV controllers and progressors, by evaluating tolerogenic and immunogenic factors in untreated HIV‐1 infected individuals. The recruited population was divided into putative elite controllers (PEC), long‐term non‐progressors (LTNP), normal progressors (NP) and fast progressors (FP). The proportion of regulatory T cells [Tregs, CD4+CD25+forkhead box P3 (FoxP3+)], programmed death (PD)‐1 and cytotoxic T lymphocyte antigen (CTLA)‐inhibitory molecules and CD40L, CD69 and Ki67 activation markers were evaluated in peripheral blood mononuclear cells (PBMC) by flow cytometry. Significant differences were found between HIV controllers and HIV progressors, with up‐regulation of Tregs, PD‐1 and CTLA‐4 and decrease of CD40L expression in progressors compared with controllers. Expression of CD40L and concentrations of interleukin (IL)‐6, CCL‐3, and CCL‐4 were significantly higher in PEC and LTNP than in NP and FP. In an attempt to convert immune signatures of progressors to those of controllers, seven agents were used to stimulate PBMC from the four cohorts. Treatment with CD40L and IL‐4 or PD‐1 antibodies in vitro were most effective in converting the immune signatures of progressors to those observed in controllers by down‐regulating Tregs and up‐regulating CD40L expression in CD4+ T cells. The conversion concept merits translation to in vivo immune control of HIV infection.

Controlled release microparticles for oral immunization

Abstract Microparticles with entrapped ovalbumin (OVA) were prepared using two different poly(lactide-co-glycolide) polymers (Resomers RG 506 and 755) with different rates of degradation and were orally administered to two groups of mice. Both groups showed enhanced serum IgG and salivary IgA antibody responses in comparison to a group of mice immunized with soluble OVA. but the level of responses induced depended on the polymer. The more rapidly degrading polymer (RG 506) was most effective for the induction of high levels of salivary IgA antibodies, while the more slowly degrading polymer (RG 755) was most effective for the induction of serum IgG antibodies.

A comparative study of stress-mediated immunological functions with the adjuvanticity of alum

Background: Alum and stress agents induce inflammasomes. The hypothesis was examined whether HSP70, a hallmark of cell stress, is involved both in alum and stress-mediated adjuvanticity. Results: Alum induced HSP70-dependent adjuvanticity as did the three stress agents. Conclusion: Inducible HSP70 is involved both in stress and alum-mediated adjuvant functions. Significance: Stress agents may provide an alternative strategy in developing novel adjuvants enhancing immunity. The efficacy of a vaccine is generally dependent on an adjuvant, which enhances the immune functions and alum has been widely used in human immunization. Alum activates the intracellular stress sensors inflammasomes, but whether these are responsible for the adjuvanticity is controversial. The objectives of this investigation were to examine the hypothesis that alum-mediated adjuvanticity is a function of stress and conversely that stress agents will elicit adjuvanticity. The investigation was carried out in BALB/c mice by SC immunization with ovalbumin (OVA) mixed with alum. This elicited inflammasomes, with significant activation of caspase 1, production of IL-1β, and adjuvanticity, demonstrated by enhancing OVA-specific serum IgG antibodies, CD4+ T cells, and proliferation. The novel finding that alum induced HSP70 suggests that stress is involved in the mechanism of adjuvanticity. This was confirmed by inhibition studies with PES (phenylethynesulfonamide), which disrupts inducible HSP70 function, and inhibited both inflammasomes and the adjuvant function. Parallel studies were pursued with an oxidative agent (sodium arsenite), K-releasing agent (Gramicidin) and a metal ionophore (dithiocarbamate). All 3 stress agents induced HSP70, inflammasomes, and the adjuvant functions. Furthermore, up-regulation of membrane associated IL-15 on DC and CD40L on T cells in the animals treated with alum or the stress agents mediate the interactions between splenic CD11c DC and CD4+ or CD8+ T cells. The results suggest that the three stress agents elicit HSP70, a hallmark of stress, as well as inflammasomes and adjuvanticity, commensurate with those of alum, which may provide an alternative strategy in developing novel adjuvants.

A recombinant human HLA-class I antigen linked to dextran elicits innate and adaptive immune responses

The objective of this study was to produce and evaluate the immunogenic potential of a recombinant HLA-class I antigen linked to dextran. The HLA-A*0201 heavy chain and beta2 microglobulin were cloned by PCR amplification of overlapping oligonucleotides and produced in E. coli. These were assembled with a CMV binding peptide motif, the HLA complex was biotinylated and bound by streptavidin coated dextran at a ratio of 24 HLA to 1 dextran molecule (termed Dextramer). Allostimulation of human PBMC in vitro and in vivo immunization of Balb c mice with the HLA-A*0201 construct elicited CD4+ and CD8+ T cell proliferative responses, IgG specific antibodies in mice and in human T cell proliferation and APOBEC3G mRNA. These adaptive and innate immune responses induced by a novel recombinant HLA construct in human cells and mice suggest their application as a potential vaccine candidate against HIV infection.

Immunization with recombinant macaque major histocompatibility complex class I and II and human immunodeficiency virus gp140 inhibits simian-human immunodeficiency virus infection in macaques.

Genetic, epidemiological and experimental evidence suggest that the major histocompatibility complex (MHC) is critical in controlling human immunodeficiency virus (HIV) infection. The objectives of this study were to determine whether novel recombinant Mamu MHC constructs would elicit protection against rectal challenge with heterologous simian-human immunodeficiency virus (SHIV) strain SF162.P4 in rhesus macaques. Mamu class I and II gene products were linked together with HIV gp140, simian immunodeficiency virus (SIV) p27 and heat-shock protein 70 to dextran. The vaccine was administered to two groups, each consisting of nine macaques, either subcutaneously (SC), or rectally and boosted by SC immunization. The controls were untreated or adjuvant-treated animals. Repetitive rectal challenges with up to ten doses of SHIV SF162.P4 showed a significant decrease in the peak and sequential viral RNA concentrations, and three macaques remained uninfected, in the nine SC-immunized animals, compared with infection in all nine controls. Macaques immunized rectally followed by SC boosters showed a less significant decrease in both sequential and peak viral loads compared with the SC-immunized animals, and all were infected following rectal challenge with SHIV SF162.P4. Plasma and mucosal IgG and IgA antibodies to Mamu class I alleles and HIV gp120, as well as to RANTES (regulated upon activation, normal T-cell expressed, and secreted; CCR5) were increased, and showed significant inverse correlations with the peak viral load. These results suggested that allo-immunization with recombinant MHC constructs linked to HIV-SIV antigens merits further investigation in preventing HIV-1 infection.

Effect of Vaginal Immunization with HIVgp140 and HSP70 on HIV-1 Replication and Innate and T Cell Adaptive Immunity in Women

ABSTRACT The international effort to prevent HIV-1 infection by vaccination has failed to develop an effective vaccine. The aim of this vaccine trial in women was to administer by the vaginal mucosal route a vaccine consisting of HIV-1 gp140 linked to the chaperone 70-kDa heat shock protein (HSP70). The primary objective was to determine the safety of the vaccine. The secondary objective was to examine HIV-1 infectivity ex vivo and innate and adaptive immunity to HIV-1. Protocol-defined female volunteers were recruited. HIV-1 CN54gp140 linked to HSP70 was administered by the vaginal route. Significant adverse reactions were not detected. HIV-1 was significantly inhibited ex vivo in postimmunization CD4+ T cells compared with preimmunization CD4+ T cells. The innate antiviral restrictive factor APOBEC3G was significantly upregulated, as were CC chemokines which induce downregulation of CCR5 in CD4+ T cells. Indeed, a significant inverse correlation between the proportion of CCR5+ T cells and the concentration of CCL-3 or CCL-5 was found. Importantly, the upregulation of APOBEC3G showed a significant inverse correlation, whereas CCR5 exhibited a trend to correlate with inhibition of HIV-1 infection (r = 0.51). Furthermore, specific CD4+ and CD8+ T cell proliferative responses were significantly increased and CD4+ T cells showed a trend to have an inverse correlation with the viral load (r = −0.60). However, HIVgp140-specific IgG or IgA antibodies were not detected. The results provide proof of concept that an innate mechanism consisting of CC chemokines, APOBEC3G, and adaptive immunity by CD4 and CD8 T cells might be involved in controlling HIV-1 infectivity following vaginal mucosal immunization in women. (This study has been registered at ClinicalTrials.gov under registration no. NCT01285141.) IMPORTANCE Vaginal immunization of women with a vaccine consisting of HIVgp140 linked to the 70-kDa heat shock protein (HSP70) elicited ex vivo significant inhibition of HIV-1 replication in postimmunization CD4+ T cells compared with that in preimmunization peripheral blood mononuclear cells. There were no significant adverse events. The vaccine induced the significant upregulation of CC chemokines and the downmodulation of CCR5 expression in CD4+ T cells, as well as an inverse correlation between them. Furthermore, the level of CCR5 expression was directly correlated with the viral load, consistent with the protective mechanism in which a decrease in CCR5 molecules on CD4+ T cells decreases HIV-1 envelope binding. Expression of the antiviral restriction factor APOBEC3G was inversely correlated with the viral load, suggesting that it may inhibit intracellular HIV-1 replication. Both CD4+ and CD8+ T cells showed HIVgp140- and HSP70-specific proliferation. A strong inverse correlation between the proportion of CC chemokine-modulated CCR5-expressing CD4+ T cells and the stimulation of CD4+ or CD8+ T cell proliferation by HIVgp140 was found, demonstrating a significant interaction between innate and adaptive immunity. This is the first clinical trial of vaginal immunization in women using only HIVgp140 and HSP70 administered by the mucosal route (3 times) in which a dual innate protective mechanism was induced and enhanced by significant adaptive CD4+ and CD8+ T cell proliferative responses.