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The importance of natural IgM antibodies in protection against infections is still emerging and these antibodies have a potential role in the maintenance of homeostasis through clearance of apoptotic bodies, complement-dependent mechanisms, inflammation and exclusion of misfolded proteins. Natural IgM act as a first line of defence against unknown hazardous factors and are present in most vertebrates. We investigated the functional capacity of anti-HIV-1 IgM monoclonal antibodies, from a combinatorial Fab library derived from healthy individuals, and evaluated their protective role in inhibiting HIV-1 in vitro when passing across the human mucosal epithelial barrier. Primary HIV-1 isolates were efficiently transmitted over the tight polarized epithelial cells when added to their apical surface. Efficient inhibition of HIV-1 transmission was achieved when anti-HIV-1 IgM monoclonal antibodies were added to the basolateral side of the cells. Two of these human IgM MoAbs had the ability to neutralize HIV and reduced infection of dendritic cells in primary cervico-vaginal tissue biopsies in vitro. This indicates a potential role of natural IgM antibodies in the reduction of HIV-1 transmission in mucosal tissues and improve our understanding of how natural IgM antibodies against a neutralizing epitope could interfere with viral transmission.

The immune response to HIV-1 virus-like particles (VLPs), presenting a clade A Ugandan gp120, has been evaluated in a mouse model by intra-nasal (i.n.) administration by a VLP+VLP homologous or a DNA+VLP heterologous prime-boost immunization protocol, including a HIV-1 DNA gp160/rev plasmid. Furthermore, the effect of the Eurocine lipid-based mucosal L3 adjuvant on the VLP immunogenicity has been assessed as well. The designed heterologous protocol is able to increase the env-specific humoral and cellular immune response, compared to the homologous protocol, which is to some extent increased by the administration of L3-adjuvanted VLP boosting dose. The anti-gag response is statistically increased in both homologous and heterologous protocols, particularly when the VLP boosting dose is adjuvanted. Immune sera from immunized animals exhibit >50% ex vivo neutralizing activity against heterologous A and B-clade viral isolates. An envelope B-cell epitope mapping shows an enhanced response against V3 epitopes all across the C2-V5 region in the heterologous prime-boost immunization strategy. The induction of humoral immunity at mucosal sites, which represents the main port of entry for the HIV-1 infection, is extremely relevant. In this framework, the DNA-VLP heterologous prime-boost protocol appears a promising preventive vaccine approach which can significantly benefit from specific mucosal adjuvants, as the Eurocine L3.

An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1-9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse's life span.

There is an urgent need for a universally effective HIV-1 vaccine, but whether a vaccine will be able to protect against HIV-1 of different clades is a significant concern. IgA from HIV-1-exposed, persistently seronegative (HEPS) subjects has been shown to neutralize HIV-1 and to block epithelial HIV-1 transcytosis, and it may target novel HIV-1 epitopes. We have tested the ability of plasma and mucosal IgA purified from HEPS subjects to neutralize HIV-1 primary isolates of different viral clades and phenotypes. IgA from two groups of HEPS subjects was tested: sex workers from Nairobi, Kenya, where clades A and D predominate, and the heterosexual partners of individuals infected by clade B virus. HIV-1-infected and low-risk uninfected individuals were included as controls. IgA purified from the blood, genital tract, and saliva of most HEPS sex workers demonstrated significant cross-clade HIV-1 neutralization, whereas a more clade-restricted pattern of neutralization was found in partners of clade B-infected individuals. IgA purified from HIV-1-infected individuals also mediated cross-clade neutralization, whereas IgA from uninfected controls lacked neutralizing activity. In conclusion, mucosal and plasma IgA from HEPS subjects neutralizes HIV-1 of different clades. This ability to induce HIV-1-specific systemic and mucosal IgA may be an important feature of an effective prophylactic HIV-1 vaccine.

Although HIV-specific cellular immune responses are found in a number of HIV highly-exposed, persistently seronegative (HEPS) cohorts, late seroconversion can occur despite pre-existing cytotoxic T lymphocytes (CTL), suggesting that a protective HIV vaccine may need to induce a broader range of HIV-specific immune responses. Low levels of HIV-specific IgA have been found in the genital tract and plasma of the majority of Nairobi HEPS sex workers and appeared to be independent of HIV-specific cellular responses. IgA purified from genital tract, saliva and plasma of most HEPS sex workers were able to neutralize infection of PBMC by a primary (NSI) clade B HIV isolate, as well as viral isolates from clades A and D, which predominate in Kenya. In addition, these IgA were able to inhibit transcytosis of infective HIV virions across a transwell model of the human mucosal epithelium in an HIV-specific manner. Preliminary work in other HEPS cohorts has suggested the recognition of different gp41 epitopes in HEPS and HIV-infected subjects. Although present at low levels, these IgA demonstrated cross-clade neutralizing activity and were able to inhibit HIV mucosal transcytosis, suggesting an important functional role in protection against HIV infection.

In order to be used as an alternative or complementary test to confirm HIV-1 infection, the efficiency of indirect immunofluorescence assay (IFA) was compared with Western blot (WB) in 362 samples from persons with high and low risk behaviour. A panel of sera with 220 WB positive, 122 WB negative and 20 WB indeterminate sera were tested by an "in house" IFA. The sensitivity of IFA was found to be 98.63% and the specificity 98.36%. Therefore, IFA appeared to be an efficient alternative method to WB, since the cost of testing by IFA is less than 10% of WB testing. We observed a direct relationship between WB protein reactivity and IFA results. In 15 samples with coincident indeterminate results for WB and IFA, antibody reactivity to p24 and gp160 presented the highest frequency. On the other hand, antibodies to viral glycoproteins were always present in IFA weak positive samples, showing their high predictive value.

Serologic assays could be useful for determining circulating subtypes in different geographic regions. A total of 175 serum samples from the same number of Argentinian HIV-infected patients from Buenos Aires and Rosario were tested against a panel of peptides representing V3 consensus subtypes A through H. A V3 peptide enzyme immunoassay was used for screening the sera. Most sera were reactive with peptides representing subtypes B (58.28%), F (13.14%), and A (8.57%). Cross-reactivity between the remainder of the peptides was observed. Genotypes of eight patients from Rosario were determined and compared with serotyping. Results showed that seven of eight genotyped patients reacted with their respective consensus B peptide and one reacted with consensus B and F. V3 peptide serology proved to be useful for determining HIV-1 clades circulating in Argentina.