Elevated levels of cytokines in mucosal secretions may function as markers of inflammatory reactions due to ongoing infection. However, limited information regarding the most efficient lechmigues and assays to be used to investigate these complex samples are available. In this study we compared four different mucosal collection device to detect nine different human cytokines in saliva and rectal wash samples spiked with standard samples of cytokines. The most reproducible and highest yields of recovery were obtained with the Wick-Cel and an experimental filter paper device, where >70% of 6/9 added cytokines were recovred (p<0.05). All four device were shown to have equal efficiency (>95% yield) to harves IgA and IgG from mucosal samples.

The immune response to HIV-1 virus-like particles (VLPs), presenting a clade A Ugandan gp 120, 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 gp 160/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 Immoral 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 beterologous 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. (c) 2007 Elsevier Ltd. All rights reserved.

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.

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.

Immunization performed intranasally with HIV-1 DNA gp160/rev plasmid with or without CCR5-DNA plasmid in a novel mucosal cationic lipid adjuvant and gp41 peptide in L3 lipid adjuvant boostered a long-lasting HIV-1 subtype A, B and C specific systemic and mucosal B- and T-cell immune memory in mice. The inclusion of the HIV co receptor CCR5 component in the vaccine may have enhanced the immunity towards HIV antigens. HIV-1 neutralizing antibodies were induced only in animals who received DNA-plasmid immunizations both in serum and in rectal mucosa. Systemic IFN-gamma secreting cell-mediated responses against the HIV-1 envelope were seen still 12 months after immunization with HIV-1 DNA and peptide if N3/L3 adjuvants were used. Peptide immunised mice responded by developing a long-tenn gp41 specific systemic non-HIV neutralizing serum IgG responses but a shorter, upto 9 months long, mucosal gp41 specific IgA immunity. Intranasal administration of low amounts of recombinant HIV-1 gag/gp120 virus-like particles (2ug rVLP gag/gp120) resulted in serum IgG/IgA, vaginal and rectal IgA against HIV-1 gag antigens at after one single immunization when the L3 adjuvant was used. Long-term serum IgG and IgA against the human and macaque CCR5 N-terminal and 2(nd) outer loop region were detectable 8 months after the last immunization in one out of two macaques.The study was designed to analyse whether HIV-1 envelope DNA and gp41 transmembrane peptides mixed in novel adjuvants for mucosal immunization could provide a broadly HIV-1 subtype recognizing long-term H]V-I envelope and/or CCR5 peptide specific immunity, similar to what has previously been reported in highly exposed persistently HIV-1 seronegative individuals.

Background: HIV-specific IgA is present in HIV-exposed uninfected individuals (EU) and neutralizes primary strains of HIV-1 in vitro.Objectives: To analyse the antigenic correlates of HIV-1 neutralization using HIV epitopes and IgA from EU and HIV-seropositive individuals.Methods: Sera from six heterosexual couples discordant for HIV serostatus, six age-matched HIV-infected subjects and six healthy controls (HC; as negative controls) were analysed. IgA binding on HIV Env recombinant proteins was assayed. Serum IgA was affinity purified on specific Env peptides and tested in HIV neutralization using resting and activated peripheral blood mononuclear cells as target. Monoclonal antibody 2F5 was used as neutralizing positive control. BALB/c mice were immunized with specific gp41 peptide and anti-sera were tested in syncytia formation and in HIV viral replication.Results: IgA of EU exclusively bound an epitope within gp41; this epitope was restricted to residues 582-588 (QARILAV) and corresponded to the leucine zip motif in the a-helical region. IgA of HIV-positive patients recognized epitopes expressed both in gp120 and gp41; these epitopes were in the N-terminal portion of the extramembrane region. Additionally, IgA of EU and antisera of QARILAV-immunized Balb/C mice blocked syncytia formation and viral replication. The dose-dependent neutralization behaviour of specific QARILAV-purified IgA was very similar to that obtained with monoclonal antibody 2F5.Conclusion: These results have important implications for the development of vaccines and therapeutical strategies against HIV infection. (C) 2002 Lippincott Williams Wilkins.

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.

OBJECTIVE:To characterize functional properties of HIV-specific IgA in samples representing both systemic and mucosal compartments of HIV-1 highly exposed persistently seronegative (HEPS) individuals.METHODS:IgA was purified from plasma and mucosal samples from HEPS individuals and tested for the ability to neutralize infection of peripheral blood mononuclear cells (PBMC) by a non-syncytium inducing HIV-1 (clade B) primary isolate. None of these individuals had measurable HIV-1-specific IgG.RESULTS:HIV-1-specific neutralizing activity of the purified IgA from plasma (n = 15), saliva (n = 15) and cervicovaginal fluid (CVF) (n = 14) were found in the majority of samples (73, 73 and 79%, respectively). In contrast, plasma, saliva and CVF samples of low-risk, uninfected HIV-seronegative individuals lacked neutralizing IgA, with the exception of two out of 34 (6%) saliva samples.CONCLUSION:Mucosal and plasma IgA from HEPS individuals can neutralize HIV-1 infection.

We investigated the immune response against a human immunodeficiency virus type 1 (HIV-1) nef DNA sequence administered epidermally in mice transgenic for the human major histocompatibility complex (MHC) class I molecule HLA-A201. Ten potential HLA-A2 binding 9-mer Nef peptides were identified by a computer-based search algorithm. By a cell surface MHC class I stabilization assay, four peptides were scored as good binders, whereas two peptides bound weakly to HLA-A2. After DNA immunization, cytotoxic T lymphocyte (CTL) responses were predominantly directed against the Nef 44-52, 81-89, and 85-93 peptides. Interestingly, the 44-52 epitope resides outside the regions of Nef where previously described CTL epitopes are clustered. Dominance among Nef-derived peptides did not strictly correlate with HLA-A2 binding, in that only one of the high-affinity binding peptides was targeted in the CTL response. The 44-52, 85-93, and 139-147 peptides also generated specific CTLs in response to peptide immunization. T helper cell proliferation was detected after stimulation with 20-mer peptides in vitro. Three Nef regions (16-35, 106-125, and 166-185) dominated the T helper cell proliferation. The implications of these results for the development of DNA-based vaccines against HIV is discussed.

HIV-1-specific IgA has been described in the genital tract and plasma of HIV-1 highly exposed, persistently seronegative (HEPS) individuals, and IgA from these sites has been shown to neutralize HIV-1. This study examines the ability of IgA isolated from HEPS individuals to inhibit transcytosis across a tight epithelial cell layer. A Transwell system was established to model HIV-1 infection across the human mucosal epithelium. The apical-basolateral transcytosis of primary HIV-1 isolates across this mucosal model was examined in the presence and the absence of IgA isolated from the genital tract, saliva, and plasma of HEPS individuals enrolled in both a sex worker cohort in Nairobi, Kenya, and a discordant couple cohort in Italy. In the absence of IgA, HIV-1 primary isolates were actively transported across the epithelial membrane and were released on the opposite side of the barrier. These transcytosed HIV-1 particles retained their ability to infect human mononuclear cells. However, IgA purified from the mucosa and plasma of HEPS individuals was able to inhibit HIV-1 transcytosis. Inhibition was seen in three of six cervicovaginal fluid samples, five of 10 saliva samples, and three of six plasma samples against at least one of the two primary HIV-1 isolates tested. IgA from low risk, healthy control subjects had no inhibitory effect on HIV-1 transcytosis. The ability of mucosal and plasma IgA to inhibit HIV-1 transcytosis across the mucosal epithelium may represent an important mechanism for protection against the sexual acquisition of HIV-1 infection in HEPS individuals.

HIV-1-specific IgA has been described in the genital tract and plasma of HIV-1 highly exposed, persistently seronegative (HEPS) individuals, and IgA from these sites has been shown to neutralize HIV-1. This study examines the ability of IgA isolated from HEPS individuals to inhibit transcytosis across a tight epithelial cell layer. A Transwell system was established to model HIV-1 infection across the human mucosal epithelium. The apical-basolateral transcytosis of primary HIV-1 isolates across this mucosal model was examined in the presence and the absence of IgA isolated from the genital tract, saliva, and plasma of HEPS individuals enrolled in both a sex worker cohort in Nairobi, Kenya, and a discordant couple cohort in Italy. In the absence of IgA, HIV-1 primary isolates were actively transported across the epithelial membrane and were released on the opposite side of the barrier. These transcytosed HIV-1 particles retained their ability to infect human mononuclear cells. However, IgA purified from the mucosa and plasma of HEPS individuals was able to inhibit HIV-1 transcytosis. Inhibition was seen in three of six cervicovaginal fluid samples, five of 10 saliva samples, and three of six plasma samples against at least one of the two primary HIV-1 isolates tested. IgA from low risk, healthy control subjects had no inhibitory effect on HIV-1 transcytosis. The ability of mucosal and plasma IgA to inhibit HIV-1 transcytosis across the mucosal epithelium may represent an important mechanism for protection against the sexual acquisition of HIV-1 infection in HEPS individuals.

An HIV-1 p24 capture enzyme linked immunosorbent assay (ELISA) was developed and used in a study of B-cell epitopes in rabbits immunised with different gag p24 antigens. Rabbits were immunised with virion HIV-1/Lai, baculovirus recombinant p24, Escherichia coli recombinant p24-15 and a mixture of synthetic peptides representing sequences of HIV-1 gag p24 protein, respectively. Five out of nine rabbits developed antibodies that could be used for an antigen capture ELISA. No significant differences in IgG titers to the whole gag protein were seen when comparing rabbits immunised with four different antigens. Three major common linear epitope regions were mapped in the rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24. The rabbit immunised with HIV-1 gag peptides had the broadest linear epitope reactive responses whereas animals immunised with E. coli recombinant antigen had the most restricted linear epitope response. The capture ELISA method thus developed using the different rabbit anti-p24 IgG preparations was shown to capture isolates from HIV-1 subtypes or clades A to G. Only rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24 developed IgG that was capable of efficiently capturing HIV-1 p24 in ELISA, indicating the importance of preparing antibodies able to recognise native or discontinuous and linear antigen configurations.

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.

To the Editor: In 1980, the human T-cell lymphotropic virus type I (HTLV-I) was first recognized as a human retrovirus. It has been considered the causative agent of adult T-cell leukemia or lymphoma and of tropical spastic paraparesis (1). This virus is endemic in several regions of Japan, central and northern South America, and Africa. An estimated 20 million people are infected with it world-wide (2). Human T-cell lymphotropic virus type II was originally isolated from two patients with hairy T cell leukemia (3). There is a wide variation in HTLV-I seroprevalence from one country to another, with the highest prevalence in Jamaica, Trinidad, and Tobago; the southern coastal regions of Colombia in South America; and the island of Kyushu in Japan (4), while HTLV-II has been shown to be endemic among Amerindian populations (5). Previous studies carried out in Buenos Aires, Argentina reported the presence of HTLV-I and HTLV-II in intravenous drug users with a prevalence of 4.2% for each retrovirus (6). Another survey performed in blood donors showed a prevalence of 0.046% and 0.023% for HTLV-I and HTLV-II, respectively (7). To determine the presence of HTLV-I/II, sera from 654 hemodialysis patients attending several hemodialysis centers in Buenos Aires, Argentina were collected during 1994. All sera specimens were screened for HTLV-I/II by particle agglutination (PA) test (Serodia HTLV-I, Fujirebio Inc, Tokyo, Japan). Reactive samples were retested by HTLV-I/II enzyme-linked immunosorbent assay (ELISA) technique (Vironostika HTLV-I Microelisa System, Organon Teknika Corporation, Durham, North Carolina, U.S.A.). Reactive and discordant results by both tests were assayed by an in-house indirect immunofluorescence assay (IFA), using an HTLV-I transformed human T-cell line (MT-2) and an HTLV-II cell line (MOT) to differentiate HTLV type I and type II (8,9). All samples were later confirmed by Western blot (WB) technique (HTLV BLOT 2.3, Diagnostic Biotechnology (Pte) Ltd., Singapore). A specimen was considered positive for HTLV-I when fulfilling the following criteria: reactivity to gag (p19 or p24), env (gp46 or rgp 46-I); and rpg 21; and for HTLV-II with gag (p24), env (rgp46-II), and rgp21. Of the 654 samples studied, 648 (99.47%) were negative; 5 (0.52%) indeterminate; and only 1 (0.10%) proved to be positive for HTLV-I. As shown in Table 1, PA test demonstrated many false-positive reactions. Although other studies show that this test is a sensitive and specific assay for the detection of HTLV-I/II, IFA results agreed with those of ELISA and WB tests. On the other hand, of the five indeterminate results by WB, four reacted with gp21. Interestingly, the other one showed a banding pattern with gp21 and rgp46-II, which could be an HTLV-II seroconversion. The finding of one patient infected with HTLV-I and a possible seroconversion for HTLV-II among the total patients studied, indicated that HTLV infection is rare in hemodialysis patients and suggests that hemodialysis is not a significant risk factor for HTLV infection. Claudia DeVito; Sandra Pampuro; Noemi Del Pino; Liliana Martinez Peralta; Osvaldo Libonatti National Reference Centre for Aids; Department of Microbiology; University of Buenos Aires; School of Medicine; Buenos Aires, Argentina