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.

In establishing preventive strategies that reduce the risk of HIV infection, mucosal immune responses induced by vaccination are of great importance, considering that HIV, like many other pathogens, is generally transmitted through the mucosa. Although the incidence of HIV-1 transmission is very low (0.0001–0.0004 in discordant couples), there is the possibility of becoming infected during each sexual act (1, 2). A high frequency of sexual encounters, different viral loads of HIV-1 carriers, and sexually transmitted diseases contribute to transmission of the virus. Inflammations and lesions in the mucosal tissues also influence HIV-1 transmission, which occurs with every genetic HIV-1 subtype or infected cell (3). To prevent infection from different pathogens, the immune system reacts quickly, producing peptides, NK cells, macrophages, and a number of Abs, such as IgM, IgG, and IgA. However, innate responses, thought to be effective in most cases, are not enough; they are unspecific and do not produce a secondary response. Therefore, specific immune responses are needed to control the spread of the virus. Mucosal microbicides are being used as a therapeutic approach (4, 5). Microbicides may acts as lubricants, protecting the mucosal surfaces, reducing the trauma, preventing sexually transmitted diseases, reducing the risk of inflammation and ulceration, and maintaining a low pH. The microbicides have been designed to target dendritic cells, reverse transcriptase, and HIV fusion in the same way as peptides based on gp 41, T-20 and T-1249 (6, 7). HIV produces a chronic infection and integrates into the host chromosomes, which indicates that prophylactic vaccination is the only way to inhibit the spread of the virus.

Many current vaccine candidates induce cellular immune responses and low levels of neutralizing Abs, meaning that those immunogens will be useful only when the infection is already established, but will not have any sterilizing effect (8, 9, 10, 11). Administration of DNA immunogens has been shown to be a potent alternative to induce cellular, although poor humoral, responses in mice and humans. Prime-booster immunization with DNA vaccine encoding the gp of herpes simplex virus (gB)3 expressed and attenuated recombinant vaccinia virus vector (rvacgB) induced mucosal and systemic responses (12). DNA constructs encoding multi-CTL epitopes from human, macaques, and mice have also been administrated i.m., enhancing HIV-specific CTL epitopes (13). Abs have been administrated to prevent HIV transmission, such as mAbs 2F5 and 2G12, which protected macaques against SHIV vaginal challenge (14).

To enhance the HIV-1-specific immune responses, immunogens are used in different combinations that target HIV attachment to the host cells. Thus, passive infusion of mAbs, which protects macaques from mucosal chimeric simian-human immunodeficiency virus (SHIV) infections, was combined with DNA immunization using IL-2 as adjuvant (15).

Because i.m. DNA immunization has been shown to induce mainly systemic immune responses, different ways of delivering the DNA have been evaluated to enhance the properties of the immune responses, mainly in the induction of neutralizing Abs, where the responses are not strong enough. Thus, nasal immunization may be the alternative to induce a broader response systemically and in distal mucosa, such as lungs, intestines, and vaginal secretions (16, 17).

We have used a heterologous vaccine strategy in which we first immunized with DNA encoding gp160/CCR5, then boosted with gp41 peptide to induce a long-lasting cellular and humoral response against HIV-1. This was based on our previous findings showing that serum and mucosal IgA from highly exposed, persistently seronegative individuals (HEPS) could neutralize primary isolates of HIV-1 (18, 19) and that mucosal IgA could neutralize HIV-1 primary isolates representing many subtypes (20). We have previously reported that secretory IgA (sIgA), the main humoral effector molecule in the mucosal immune system has a role in inhibiting HIV-1 epithelial transcytosis (21). It has recently been shown that IgA of HEPS recognized an epitope located on the gp41 protein that differs from the IgA epitope recognized by HIV-infected individuals restricted to aa 581–584 (LQAR), which corresponds to the conserved coiled-coil pocket in the α helix region of gp41 (22). Previous studies have also shown that certain HEPS individuals may develop Abs against the HIV-1 non-syncytium-inducing (NSI) phenotype coreceptor CCR5 (23). The protective capacity of Abs directed against this coreceptor has thus been investigated as a vaccine approach with interesting possibilities in vivo. Considering that experimental approaches inducing long-term mucosal immune responses may be necessary, we evaluated the immunogenicity of a peptide representing the gp41 coiled-coil pocket region in mice. We also evaluated whether these immune responses can be enhanced by combining HIV-1 rgp160/Rev DNA, CCR5 DNA, and CCR5 peptides with other well-conserved gp41 epitopes of the envelope from HIV-1 clades A, B, C, and D (24). A spectrum of specific epitopes from subtypes A–D provides both broad HIV-1 clade immunity combined with autoantibodies against the prominent coreceptor CCR5. We hypothesized that this approach would result in a more robust and long-lasting neutralizing mucosal immunity able to resist the development of escape mutations, which is so commonly seen with the HIV-1 virus.

Female 10- to 12-wk-old C57BL/6 mice of the H-2b haplotype were immunized intranasally with HIV-1 rgp160BaL DNA, HIV-1 Rev/Lai DNA, human CCR5 DNA, gp41/MN coiled-coil (aa 578–591, GIKQLQARVLAVERY), and gp41 subtype A–D peptides (aa 661–675, MN, NEQLLELDKWASLWN, A/92UG31; aa 652–665, EKDLLALDKWANLWN, C/92BR025; aa 651–665, NEQDLLALDSWNLWN, D/92UG021; aa 643–657, EQELLKLDQWASLWN), E/93TH976 aa 643–662, DQQDRNEKDLLLDKWASLW peptides, and peptides representing the human second CCR5 coreceptor (aa 168–182, FTRSQKEGLHYTCSSHFPYS) region (Hybaid T-Peptides, Ulm, Germany). All animals were immunized twice at a 2-mo interval, and the animal experiments were repeated twice. The different immunization combinations used are described in Table I. All DNA plasmids contained the CMV immediate-early promoter for gene expression and the kanamycin resistance gene. Eight control mice were included in the experiments. The intranasal booster immunization was performed with peptides (10 μg/peptide) emulsified in mono-oleate/fatty acid (named L3) as previously described (25).

Table I.

Immunization schedule of micea

Mouse GroupPrimary Immunization/Booster Immunization
rgp160/rev DNACCR5-DNAgp41 ELDKWAS peptidegp41 coil peptideCCR5 second loop peptide
+/− +/− −/+ −/+ −/+ 
+/+ −/− −/+ −/+ −/− 
−/− −/− +/+ +/+ +/+ 
−/− −/− −/− −/− +/+ 
−/− −/− −/− −/− −/− 
6 Reversed group 1 −/+ −/+ +/− +/− +/− 
Mouse GroupPrimary Immunization/Booster Immunization
rgp160/rev DNACCR5-DNAgp41 ELDKWAS peptidegp41 coil peptideCCR5 second loop peptide
+/− +/− −/+ −/+ −/+ 
+/+ −/− −/+ −/+ −/− 
−/− −/− +/+ +/+ +/+ 
−/− −/− −/− −/− +/+ 
−/− −/− −/− −/− −/− 
6 Reversed group 1 −/+ −/+ +/− +/− +/− 
a

Six mice were used in each group except in the control group 5 controls receiving saline which contained eight mice. Groups 1, 2, 3, 5, and 6 were repeated twice. The adjuvant monooleate/oleate L3 (groups 1, 2, 3, and 4) was used for the booster immunizations with peptides. DNA immunizations: 25 μg/plasmid; peptide immunizations: 10 μg/peptide.

This study was performed with ethical permission from the regional ethical committee (Stockholm Nord).

Blood obtained by retro-orbital bleedings, vaginal and lung lavages, and feces were collected at 1- to 3-mo intervals from each mouse. Serum was stored at −20°C until used. Mucosal samples were collected as previously described (16, 17, 26). Briefly, the vaginal and lung lavages were collected in PBS with protease inhibitors (Sigma-Aldrich, St. Louis, MO) and stored at −70°C before use. Feces were weighed and solubilized in PBS (0.1 g/ml) with protease inhibitors (1 mg/ml; Sigma-Aldrich). The debris was removed by centrifugation, and the supernatant was stored at −70°C. When mice were killed, vaginal, lung, and intestinal lavages were collected as previously shown (16, 17).

The peptides corresponding to the gp41-neutralizing epitope synthesized were aa 661–675/ELDKWAS (Hybaid T-Peptides), representing clades A/(92UG31), B/(MN), C/(92BR25), and D/(92UG21), and a gp41 clade B/(MN) peptide (Hybrid T-Peptides) located between aa 578–595 peptides, representing the human and simian CCR5 N-terminal region and second loop aa 168–182. We also synthesized aa 178–192 (Hybaid T-Peptides) and aa 302–318 from gp120 V3 loop/clade B. The HIV-1 Lai Rev peptide synthesized, representing aa 65–84, was used as a negative control. All peptides mentioned were produced using solid phase F-moc chemistry (27).

Ninety-six-well plates (Maxisorp; Nunc, Copenhagen, Denmark) were coated with clade A–D gp41 ELDKWAS peptide, gp41 coiled-coil peptide (QLQARVL) peptide, gp120 V3/MN (IHIGPGRAFV), and the human CCR5 second-loop peptides. All peptides were solubilized in 0.1 M NaHCO3 buffer (pH 9.5–9.6) at a coating concentration of 10 μg/ml and added at 100 μl/well. Plates were stored overnight at room temperature and for a minimum of 24 h at 4°C. Mouse sera were diluted in PBS (pH 7.4) with 0.5% BSA (Roche, Mannheim, Germany) and 0.05% Tween 20 (Sigma-Aldrich), and 100 μl of dilutions of 1/100, 1/200, 1/400, and 1/800 were added to each well and incubated at 37°C for 90 min.

Mucosal samples were tested as previously described (16, 17). HRP-conjugated anti-mouse IgG (Bio-Rad, Richmond, CA; dilution, 1/2000) or anti-mouse IgA (Southern Biotechnology Associates, Birmingham, AL; dilution, 1/1000) was added at 100 μl/well and incubated for 2 h at 37°C, and 2 mg/ml ophenylenediamine in 0.05 M sodium citric acid, pH 5.5, with 0.003% H2O2 were added as substrate at 100 μl/well. After a 30-min incubation period, the reaction was stopped by adding 100 μl/well 2.5 M H2SO4, and optical densities were measured at 490 nm. The positive control used was a human HIV-IgG pool collected from HIV-1-infected Ugandan patients, the Kabi 62 serum, mAbs against the gp41 ELDKWAS epitope mAb 2F5 (28) (donated by Dr. H. Katinger), anti-gp120 V3 mAb F58/H3 (29), and mAb 2D7 directed against the second CCR5 external loop region (Coulter Pharmaceuticals, Palo Alto, CA). Sera from vaginal secretions and feces from preimmunized animals were used as negative controls.

The secretions collected from lungs and intestines were used to isolate and analyze the HIV-1-specific IgA content. The Kaptive IgA/IgE reagents (Biotech IgG, Copenhagen, Denmark) were purchased and used as recommended by the manufacturer. IgA quantities were determined with an in-house murine IgA capture ELISA, and a commercial murine IgA (1 mg/ml; Sigma-Aldrich) was used for preparing a standard curve. Briefly, the purified IgA and the standard IgA were diluted in PBS with 5% fat-free dry milk and 0.05% Tween 20 at 10-fold serial dilutions. One hundred microliters per dilution was added to a 96-microwell plate precoated with rabbit anti-murine IgA (Dakopatts, Sollentuna, Sweden) and incubated at 37°C for 1 h. The plates were washed four times with saline and 0.05% Tween 20 before 100 μl of HRP-conjugated goat anti-murine IgA was added to each well. After 1-h incubation at 37°C, plates were washed as previously described, and the presence of bound conjugate was detected using o-phenylenediamine in 0.05 M sodium-citric acid, activated with 0.03% H2O2 as substrate. The substrate reaction was terminated with 100 μl/well of 2.5 M H2SO4, and absorbance was measured at OD490. The amounts of IgA in the mouse samples were determined by comparing the OD values of the test samples with that of the IgA standard with known IgA concentration.

Serum and IgA purified from feces from preimmunized and immunized mice were tested for the presence of neutralizing activity. Mouse sera were heat-inactivated (56°C for 30 min) and serially diluted at 3-fold dilutions, starting at 1/20. Virus aliquots of the dual-tropic T cell line-adapted HIV-1 SF2 strain and the primary NSI/CCR5 tropic clade B isolate 6920 (19) were diluted in quadruplicate 4-fold steps in medium supplemented with 10% inactivated FCS (Invitrogen Life Technologies, Paisley, U.K.), 10 IU/ml IL-2 (Amersham Biosciences, Little Chalfont, U.K.), 50 μg/ml streptomycin, and 50 IU penicillin (Invitrogen Life Technologies). Seventy-five microliters of each virus dilution and 75 μl of each serum dilution were incubated in duplicate for 1 h at 37°C in round-bottom culture plates (Nunc). Seventy-five microliters with 1 × 106/ml PHA-stimulated PBMC were added to each well. After 16- to 18-h incubation at 37°C, the cells were washed with RPMI 1640, and 200 μl of fresh medium was added to each well. Every 3 days, 100 μl of medium was changed. After 6–7 days, 100 μl of supernatant from each well was collected, and virus production was measured in a p24 Ag capture ELISA as previously described (30). An HIV-1-positive serum pool (HIVIG) and the human mAb 2F5, specific for the gp41 ELDKWAS epitope, were used as a positive control. Neutralization was defined as the sample titer resulting in 50 and 90% reduction of p24 Ag in the supernatant compared with p24 Ag content when the virus was incubated in the presence of HIV Ab from negative serum. All assays were repeated at least twice.

HIV-1-specific IgA neutralization activity was performed as previously described (28). In brief, purified fecal IgA from control or immunized mice was incubated at 37°C for 1 h with 30–50 tissue-culture infectious doses resulting in 50% infected culture wells of cell-free virus HIV-1SF2 (T cell line-adapted subtype B isolate) or with the primary HIV subtype B isolate 6920 in a 200-μl volume in duplicate 96-well cell culture plates (Nunc, Aarhus, Denmark).

For analysis of HIV-specific B cell responses, 1 × 105 spleen cells were cultured in 200 μl of RPMI 1640 supplemented with 5% inactivated FCS at 37°C in 96-well, flat-bottom plates (Nunc) with rgp160 (1 μg/ml) or peptides (10 μg/ml) for 72 h. After washing, Ag-specific Igs were measured by ELISA as described above (14, 26). A positive reactivity was considered if the ELISA substrate absorbance was higher than the mean value OD ± 2 SD of the negative control. T cell proliferation assay was performed as previously described (4, 11).

Statistical comparisons between the groups were performed using the nonparametric Mann-Whitney U test. A significant difference was considered at a value of p < 0.05. A one-way ANOVA nonparametric test was performed using PRISM version 4.0a (GraphPad, San Diego, CA) for MacIntosh (OS 9; Apple Computers, Redmond, WA) for comparisons of medians between groups at p < 0.05 and p < 0.001 levels when comparing fecal and vaginal IgA titers.

Serum HIV-specific IgG responses against gp41, gp120, gp160, and CCR5 were measured 1 year after postboost immunization. The geometric mean titers 6 and 12 mo after intranasal prime and booster immunizations are given in Table II. High Ab titers were seen against all gp41 peptides. HIV-specific IgG persisted over 12 mo, and no significant differences were detected among groups 1, 2, and 3 (p > 0.05) at 6 or 12 mo or in the variations in serum IgG titer at the two time points measured. The lowest IgG titers were detected against gp41 coiled-coil peptide, gp120 V3, gp160Lai, and CCR5. Mice immunized only with CCR5 peptides (group 4) had equally high serum IgG Ab titers to CCR5 as mice from group 1, which also received CCR5/DNA, whereas the control mice (group 5) had low or undetectable IgG titers (<100) against all HIV-1 Ags used. Among the groups of mice in which immunizations were repeated, the mice primarily immunized with gp41/CCR5 peptides in adjuvant and 2 mo later immunized with gp160/CCR5 DNA had Ab responses similar to those in the group 3 mice receiving peptides only (not shown).

Table II.

Serum Ab levels following intranasal immunizationa

GroupTime (mo)Geometric Mean IgG Titers of Clade Agna CCR5 loop aa 168–185
B gp41 MN aa 661–675A gp41 UG31 aa 652–665C gp41 BR25 aa 651–665D gp41 UG21 aa 643–657B gp41 coil aa 578–592B gp120 V3 aa 303–322B rgp160Lai
4,525 5,382 3,805 2,691 336 800 336 1,345 
 12 3,805 3,200 2,691 1,345 336 336 566 1,903 
3,676 4,850 4,222 4,222 400 696 459 <100 
 12 1,832 3,200 2,111 1,838 200 132 400 <100 
2,786 2,786 3,676 1,600 264 528 606 115 
 12 2,111 1,600 1,600 919 174 100 400 159 
<100 <100 <100 <100 <100 <100 <100 770 
 12 <100 <100 <100 <100 <100 <100 <100 696 
<100 <100 <100 <100 <100 <100 <100 <100 
 12 <100 <100 <100 <100 <100 <100 <100 <100 
mAb 2F5 Positive controlb 12,400 1,440 680 <100 <100 <100 57,600 <100 
GroupTime (mo)Geometric Mean IgG Titers of Clade Agna CCR5 loop aa 168–185
B gp41 MN aa 661–675A gp41 UG31 aa 652–665C gp41 BR25 aa 651–665D gp41 UG21 aa 643–657B gp41 coil aa 578–592B gp120 V3 aa 303–322B rgp160Lai
4,525 5,382 3,805 2,691 336 800 336 1,345 
 12 3,805 3,200 2,691 1,345 336 336 566 1,903 
3,676 4,850 4,222 4,222 400 696 459 <100 
 12 1,832 3,200 2,111 1,838 200 132 400 <100 
2,786 2,786 3,676 1,600 264 528 606 115 
 12 2,111 1,600 1,600 919 174 100 400 159 
<100 <100 <100 <100 <100 <100 <100 770 
 12 <100 <100 <100 <100 <100 <100 <100 696 
<100 <100 <100 <100 <100 <100 <100 <100 
 12 <100 <100 <100 <100 <100 <100 <100 <100 
mAb 2F5 Positive controlb 12,400 1,440 680 <100 <100 <100 57,600 <100 
a

IgG titers against HIV-1 gp41, gp120, rgp160, and CCR5 representing Ags 6 and 12 mo post-boost immunization. rgp160 = recombinant gp160, na, not applicable.

b

Positive IgG control 5 μg/ml (HRP-anti-IgG conjugate used). Statistical analysis using Mann-Whitney U nonparametric test were used to compare differences between groups and p = 0.05 was considered significant. No significant differences were seen when comparisons were performed between relevant groups.

Serum IgA titers against HIV-1 and CCR5 were detected as late as 1 year postboost immunization (Table III). No significant geometric differences in the mean of IgA Ab titers (p > 0.05) were detected among groups 1, 2, and 3 against the gp41 clade A, B, and C peptides, whereas the IgA titers against clade D gp41 were lower in group 3 (p < 0.05). In addition, group 3 lacked detectable IgA Abs against the V3 loop region. The HIV-specific IgA titers were, in general, 10-fold lower than the IgG titers. Among the mice in group 4, the highest IgA titers were seen against the CCR5 peptide. However, the mean Ab titers of this group were not significantly different from the titers obtained in group 1 (p = 0.42). In the negative control (group 5), HIV-specific IgA responses could not be detected against any of the Ags used.

Table III.

Serum IgA titers against HIV-1 gp41, gp120, and CCR5 representing Ags 6 and 12 mo post-boost immunizationa

GroupTime (mo)Geometric Mean IgA Titers of Clade Agna CCR5 loop aa 168–185
B gp41 MN aa 661–675A gp41 UG31 aa 652–665C gp41 BR25 aa 651–665D gp41 UG21 aa 643–657B gp41 coil aa 578–592B gp120 V3 aa 303–322B rgp160Lai
200 598 400 100 400 390 420 370 
 12 200 800 336 100 425 200 275 350 
303 303 303 132 303 225 160 <100 
 12 303 303 264 152 303 174 152 <100 
159 200 200 100 264 <100 200 440 
 12 159 152 174 100 <100 <100 180 160 
<100 <100 <100 <100 <100 <100 <100 790 
 12 <100 <100 <100 <100 <100 <100 <100 300 
<100 <100 <100 <100 <100 <100 <100 <100 
 12 <100 <100 <100 <100 <100 <100 <100 <100 
mAb 2F5b Positive control 10,900 1,560 990 120 <100 <100 65,000 <100 
GroupTime (mo)Geometric Mean IgA Titers of Clade Agna CCR5 loop aa 168–185
B gp41 MN aa 661–675A gp41 UG31 aa 652–665C gp41 BR25 aa 651–665D gp41 UG21 aa 643–657B gp41 coil aa 578–592B gp120 V3 aa 303–322B rgp160Lai
200 598 400 100 400 390 420 370 
 12 200 800 336 100 425 200 275 350 
303 303 303 132 303 225 160 <100 
 12 303 303 264 152 303 174 152 <100 
159 200 200 100 264 <100 200 440 
 12 159 152 174 100 <100 <100 180 160 
<100 <100 <100 <100 <100 <100 <100 790 
 12 <100 <100 <100 <100 <100 <100 <100 300 
<100 <100 <100 <100 <100 <100 <100 <100 
 12 <100 <100 <100 <100 <100 <100 <100 <100 
mAb 2F5b Positive control 10,900 1,560 990 120 <100 <100 65,000 <100 
a

na = not applicable; rgp160 = recombinant gp160 Ag.

b

Positive IgG control 5 μg/ml (HRP-anti-IgG conjugate used). Statistical analysis using Mann-Whitney U nonparametric test were used to compare differences between groups and p = 0.05 was considered significant. No significant differences were seen when comparisons were performed between relevant groups.

We analyzed the reactivity of IgA from feces against gp41 ELDKWAS peptides representing clades A, B, C, D, and E as well as CCR5. Fig. 1 shows the median IgA ELISA Ab reactivity at two different time points before and after the second immunization in groups 1–5. Animals from group 1 developed IgA against clades A, B, C, and D and CCR5 after the second booster, and the highest reactivity was detected against the CCR5 peptide (Fig. 1). None of the animals developed IgA against the control Rev peptide. When mice were killed at 12 mo follow-up (Table IV), HIV-specific IgA against gp41 ELDKWAS representing subtypes A, B, and C were detected in the small intestine of all mice from group 1. This HIV-specific reactivity was also detectable against the gp41 coiled-coil peptide. Furthermore, at 12 mo, five of six mice had intestinal IgA reactive with gp160, and four of six mice had intestinal IgA reactive against the CCR5 second-loop peptide.

FIGURE 1.

A–E, Median fecal IgA ELISA reactivity in mouse groups 1, 2, 3, 4, and 5 at two different time points, 3 wk after the primary immunization and 4 wk after the second immunization. Fecal IgA titers are shown against the gp41 peptides representing clades A, B, D, and E and the CCR5 second-loop peptide. All boxed figures show the median IgA titers and the 25th and 75th quartiles of group 1–5 mice. Significant differences between the control group 5 and the other groups at the p < 0.001 or p < 0.05 level are indicated above the bars (ns, nonsignificant difference). Values for determining cutoff limits were determined by testing fecal pellet washes from preimmunization animals by ELISA. Mean background ELISA absorbance values ± 2 SD ranged from between 0.095 and 0.150 in the various peptide Ags. A, Against gp41A-EKDLLALDKWANLWN peptide; B, against gp41B NEQLLELDKWASLWN peptide; C, against gp41D-EQELLKLDQWASLWN peptide; D, against gp41 E-RNEKDLLLDKWASLW peptide; E, and against the second-loop human CCR5 peptide (amino acids FTRSQKEGLHYTCSSHFPYS).

FIGURE 1.

A–E, Median fecal IgA ELISA reactivity in mouse groups 1, 2, 3, 4, and 5 at two different time points, 3 wk after the primary immunization and 4 wk after the second immunization. Fecal IgA titers are shown against the gp41 peptides representing clades A, B, D, and E and the CCR5 second-loop peptide. All boxed figures show the median IgA titers and the 25th and 75th quartiles of group 1–5 mice. Significant differences between the control group 5 and the other groups at the p < 0.001 or p < 0.05 level are indicated above the bars (ns, nonsignificant difference). Values for determining cutoff limits were determined by testing fecal pellet washes from preimmunization animals by ELISA. Mean background ELISA absorbance values ± 2 SD ranged from between 0.095 and 0.150 in the various peptide Ags. A, Against gp41A-EKDLLALDKWANLWN peptide; B, against gp41B NEQLLELDKWASLWN peptide; C, against gp41D-EQELLKLDQWASLWN peptide; D, against gp41 E-RNEKDLLLDKWASLW peptide; E, and against the second-loop human CCR5 peptide (amino acids FTRSQKEGLHYTCSSHFPYS).

Close modal
Table IV.

Long-term HIV- and CCR5 Ag-specific IgA responses in lung and small intestinal washes 12 mos post-boostera

GroupSamplebgp41 ELDKWAS IgA Peptide Titers/CladeCCR5 PeptideIgA Concn (μg/ml)
gp41/Agp41/Bgp41/Cgp41 Coil/Brgp160
Lung 60 16 50 18 16 15 18 ± 6 
 Intestine 46 46 80 90 50 80 16 12 30 30 20 57 ± 11 
Lung <2 <2 <2 <2 26 ± 9 
 Intestine 12 12 20 28 16 16 <4 <4 <4 86 ± 19 
Lung <2 <2 10 10 <2 <2 <2 <2 <2 16,6 ± 9 
 Intestine 20 28 <4 <4 <4 <4 <4 86 ± 27 
Lung <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 19,2 ± 8 
 Intestine <4 <4 <4 <4 <4 <4 <4 <4 <4 16 78 ± 11 
Lung <2 <2 <2 <2 <2 <2 <2 <2 <2 16 33 ± 16 
 Intestine <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 48 ± 8 
Positive controlsc              
 mAb2F5  1,490  12,000  710  <100  61,000  <100  
 HIVIG  4,900  3,900  6,800  2,900  590,000  140  
 mAb2D7  nt  nt  nt  nt  <100  220  
Negative control              
 Mouse sera  <50  <50  <50  <50  <50  <50  
GroupSamplebgp41 ELDKWAS IgA Peptide Titers/CladeCCR5 PeptideIgA Concn (μg/ml)
gp41/Agp41/Bgp41/Cgp41 Coil/Brgp160
Lung 60 16 50 18 16 15 18 ± 6 
 Intestine 46 46 80 90 50 80 16 12 30 30 20 57 ± 11 
Lung <2 <2 <2 <2 26 ± 9 
 Intestine 12 12 20 28 16 16 <4 <4 <4 86 ± 19 
Lung <2 <2 10 10 <2 <2 <2 <2 <2 16,6 ± 9 
 Intestine 20 28 <4 <4 <4 <4 <4 86 ± 27 
Lung <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 19,2 ± 8 
 Intestine <4 <4 <4 <4 <4 <4 <4 <4 <4 16 78 ± 11 
Lung <2 <2 <2 <2 <2 <2 <2 <2 <2 16 33 ± 16 
 Intestine <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 <4 48 ± 8 
Positive controlsc              
 mAb2F5  1,490  12,000  710  <100  61,000  <100  
 HIVIG  4,900  3,900  6,800  2,900  590,000  140  
 mAb2D7  nt  nt  nt  nt  <100  220  
Negative control              
 Mouse sera  <50  <50  <50  <50  <50  <50  
a

Samples collected from the lung were tested at dilution 1/2–1/118 and intestinal washes at dilutions 1/4–1/256 at 2-fold serial serum dilutions. Sample IgA titer cut-off was calculated from mean absorbance values of negative control mice plus two SD. nt, not tested.

b

Two IgA pools per mouse group were prepared and tested.

c

Positive controls used were human IgG mAb and HIVIgG (HRP-anti-human IgG conjugate used) end-point titers are shown.

HIV-1-specific IgA Abs in feces were also found in group 2 (Fig. 1). After the booster immunization, all mice developed IgA Abs against gp41 peptides representing clades A, B, C, and D, whereas in half (three of six) of the animals, the highest reactivity was detected against gp41 ELDKWAS clade B. After 12 mo (Table IV), IgA in the small intestine reacted with gp41 ELDKWAS peptides of clade A in all six mice, against clade B in five of six mice, against clade C in four of six mice, against the gp41 coil peptide in one of six mice, and against gp160 in five of six mice. None of the animals from group 2 developed IgA Abs against CCR5 or Rev peptides.

Low IgA Ab responses were seen in group 3, in which mice were immunized with gp41 ELDKWAS, CCR5, and gp41 coiled-coil peptides (Fig. 1). In this group, the highest reactivities were detected only against clade B and CCR5 peptides. When studying the intestinal IgA responses 12 mo after the booster, clade A gp41 ELDKWAS was recognized by three of six mice, that against clade C was recognized by one of six, and no responses against the gp41 coil peptide, gp160, CCR5, or Rev peptide were found. In mice primarily immunized with gp41/CCR5 peptides and boosted with HIV-gp160/CCR5 DNA, the fecal IgA reaction pattern did not differ from the group 3 mice who were given peptides only (not shown).

In group 4 mice, IgA reactivity in feces with the CCR5 second-loop peptide was seen in all six animals. After 12 mo of follow-up, all six mice had detectable IgA in the small intestine toward the CCR5 peptide without reacting with tested HIV-1 Ags. No Ab reactivity was seen in group 5 immunized with saline (Fig. 1). The total IgA amount in fecal samples ranged between 280 and 418 μg/ml; in the intestinal washes it ranged between 48 and 92 μg/ml. No significant differences were found among mice from the five studied groups.

Fig. 2 shows the median anti-HIV and CCR5 peptide IgA Ab reactivity in vaginal secretions 3–12 mo postbooster in four immunized groups and one control group of mice. Significantly higher reactivities in group 1 were detected against all HIV gp41 and CCR5 Ags compared with group 4 or 5. When studying the kinetics of vaginal HIV-specific IgA reactivity, long-term immunity (9 mo) was seen against the gp41 peptides. By 12 mo after the peptide booster, HIV-specific IgA in vaginal samples against the gp41 ELDKWAS and the gp41 coiled-coil LQAR peptides were seen. In group 2, all mice developed IgA Abs against all Ags except the CCR5 peptide, although the responses decreased with time more rapidly than in group 1 (Fig. 2).

FIGURE 2.

A–E, Kinetics of vaginal wash IgA ELISA reactivity against gp41 peptides representing HIV-1 clades A, B, and C. The median IgA titer reactivity is shown at four time points (3, 6, 9 and 12 mo after the second immunization) in groups 1–5. The boxes show the median vaginal IgA Ab titers with the 25th and 75th quartiles from four HIV Ag-immunized mouse groups (groups 1–3) and the control mice (group 5) Values for determining cutoff limits were performed by testing vaginal washes from preimmunization animals by ELISA. Mean background ELISA absorbance values ± 2 SD ranged from between 0.028 and 0.50 in the various peptide Ags. Significant differences at the p < 0.001 or p < 0.05 level (ns, nonsignificant difference) between control group 5 animals and the other groups are indicated at the top of the bars. Statistics were performed using the one-way ANOVA nonparametric analysis. A, Against the HIV-1 gp41 clade A representing peptide (aa EKDLLALDKWANLWN); B, against the HIV-1 gp41 clade B representing peptide (aa NEQLLELDKWASLWN; C, against the HIV-1 gp41 clade C representing peptide (aa NEQDLLALDSWNLWN); D, against the HIV-1 gp41 coiled-coil peptide clade B representing peptide (aa GIKQLQARVLAVERY); E, against the CCR5 second-loop peptide representing the human CCR5 HIV-1 coreceptor (aa FTRSQKEGLHYTCSSHFPYS).

FIGURE 2.

A–E, Kinetics of vaginal wash IgA ELISA reactivity against gp41 peptides representing HIV-1 clades A, B, and C. The median IgA titer reactivity is shown at four time points (3, 6, 9 and 12 mo after the second immunization) in groups 1–5. The boxes show the median vaginal IgA Ab titers with the 25th and 75th quartiles from four HIV Ag-immunized mouse groups (groups 1–3) and the control mice (group 5) Values for determining cutoff limits were performed by testing vaginal washes from preimmunization animals by ELISA. Mean background ELISA absorbance values ± 2 SD ranged from between 0.028 and 0.50 in the various peptide Ags. Significant differences at the p < 0.001 or p < 0.05 level (ns, nonsignificant difference) between control group 5 animals and the other groups are indicated at the top of the bars. Statistics were performed using the one-way ANOVA nonparametric analysis. A, Against the HIV-1 gp41 clade A representing peptide (aa EKDLLALDKWANLWN); B, against the HIV-1 gp41 clade B representing peptide (aa NEQLLELDKWASLWN; C, against the HIV-1 gp41 clade C representing peptide (aa NEQDLLALDSWNLWN); D, against the HIV-1 gp41 coiled-coil peptide clade B representing peptide (aa GIKQLQARVLAVERY); E, against the CCR5 second-loop peptide representing the human CCR5 HIV-1 coreceptor (aa FTRSQKEGLHYTCSSHFPYS).

Close modal

One of five mice in group 3 developed vaginal IgA Abs against clade C and CCR5 peptide after the first immunization. However, the group median reactivity was low or undetectable against all Ags at most time points.

In group 4, CCR5 peptide-specific vaginal IgA response was seen at 1–9 mo postbooster immunization, but did not differ significantly from group 1 vaginal IgA titers. Reactivity at later time points and against the other Ags was undetectable (Fig. 2 E). The amount of sample that can be collected from each mouse is small (100 μl/sample/occasion), and for this reason the samples were pooled for determining IgA amounts within each group (the pools were prepared from three mice). Because total IgA responses were analyzed from a pool of vaginal wash samples, the amounts of IgA measured in the preimmunized animals (3.3–8.1 μg/ml) did not significantly differ from the IgA amounts detected in the postimmunization samples (2.7–11.5 μg/ml).

Lung wash IgA was collected 12 mo postbooster immunization (Table IV). To obtain sample amounts large enough to analyze IgA reactivity against several HIV-1 and CCR5 peptides, two pools of lung washes were prepared from three mice per study group. The strongest reactivities against the gp41 ELDKWAS epitopes were seen with samples from group 1 mice. Group 2 mouse lung IgA reacted with gp41 ELDKWAS peptides from clades B and C, although at low titers (IgA titer range, 2–8). Group 3 had lung IgA against gp41 ELDKWAS peptides from clades B and C only.

The CCR5 peptide-immunized group 4 had CCR5 peptide-specific IgA only in lungs (IgA titer, 10–16). The amount of IgA in lung secretions ranged between 5.5 and 33 μg/ml samples in the various groups.

Three groups of mice (1, 2, 3) had similarly high frequencies of spleen and inguinal lymph nodes IgG and IgA/B cell responders against HIV-1 Ags, and two groups (1 and 4) had high frequencies of IgA responders against the CCR5 loop peptide at 12 mo after booster immunization. In the control group 5, one of 10 mice reacted with the CCR5 peptide, whereas there was no response against the other peptides used (not shown). At 12 mo, HIV Ag-specific in vitro T cell proliferative responses were detectable only in the DNA HIV-1 gp160 animals (groups 1 and 2). Low amounts of IFN-γ were detected in gp120 V3 peptide-stimulated spleen cells of mice from groups 1 and 2 (45–150 pg/ml; not shown).

We evaluated the ability of serum to neutralize one laboratory-adapted strain and a primary HIV-1 isolate (Figs. 3 and 4). All three sera tested neutralized the HIV-1/SF2 isolates, with 80% neutralizing titers ranging between 20 and 80 (1, 2, 3), of which the two best animals per group are shown in Figs. 3 and 4. When serum was tested for neutralization responses, all three sera in group 1 neutralized a primary HIV-1 isolate exemplified by the two best-neutralizing animal sera in Fig. 4. In group 2, three of four sera neutralized a primary HIV-1 isolate with 80% neutralizing titers ranging between 20 and 80 (Fig. 4). Sera from mice in groups 3 and 5 and mice primed with gp41/CCR5 peptides followed by an HIV-1 gp160/CCR5 DNA booster lacked detectable HIV-1-neutralizing activity (Figs. 3 and 4). The HIV-1-neutralizing sera were absorbed with HIV-1-infected or uninfected cells before repeating the HIV-1 neutralization assays. This resulted in loss of HIV-1-neutralizing activity when HIV-1-infected cells were used (Fig. 5). When uninfected cells were used to absorb serum, a slight decrease in the serum neutralization activity was seen in the plasmid DNA-primed mice (groups 1 and 2; group 2 not shown), and a complete loss of neutralizing activity was found among the CCR5 peptide-immunized mice (group 4).

FIGURE 3.

HIV-1 SF2 SI-phenotype neutralization activity of two representative mouse sera from groups 1–5 collected 9 mo after the second immunization. Sera collected 6 mo postbooster immunization from two representative mice primarily immunized with gp41/CCR5 peptides in L3 adjuvant and boosted with HIV-1 gp160/CCR5 DNA intranasally, referred to as Reversed Group 1 mice, are shown. Mouse group and numbers of individual mice are presented in Table I. A >50% neutralization was considered positive.

FIGURE 3.

HIV-1 SF2 SI-phenotype neutralization activity of two representative mouse sera from groups 1–5 collected 9 mo after the second immunization. Sera collected 6 mo postbooster immunization from two representative mice primarily immunized with gp41/CCR5 peptides in L3 adjuvant and boosted with HIV-1 gp160/CCR5 DNA intranasally, referred to as Reversed Group 1 mice, are shown. Mouse group and numbers of individual mice are presented in Table I. A >50% neutralization was considered positive.

Close modal
FIGURE 4.

HIV-1 6920 NSI phenotype primary isolate neutralization activity of two representative mouse sera from groups 1–5 collected 9 mo after the second immunization. Sera collected from two representative mice primary immunized with gp41/CCR5 peptides in L3 adjuvant and boosted with HIV-1 gp160/CCR5 DNA intranasally, referred to as Reversed Group 1 mice, are shown. Mouse group and numbers of individual mice are presented in Table I. A >50% neutralization was considered positive.

FIGURE 4.

HIV-1 6920 NSI phenotype primary isolate neutralization activity of two representative mouse sera from groups 1–5 collected 9 mo after the second immunization. Sera collected from two representative mice primary immunized with gp41/CCR5 peptides in L3 adjuvant and boosted with HIV-1 gp160/CCR5 DNA intranasally, referred to as Reversed Group 1 mice, are shown. Mouse group and numbers of individual mice are presented in Table I. A >50% neutralization was considered positive.

Close modal
FIGURE 5.

HIV-1 6920 NSI primary isolate-neutralizing activity with pooled sera collected 9 mo after booster immunization from group 1 (A) and from group 4 (B) before and after absorption with uninfected and HIV-1 6920-infected U937 cells.

FIGURE 5.

HIV-1 6920 NSI primary isolate-neutralizing activity with pooled sera collected 9 mo after booster immunization from group 1 (A) and from group 4 (B) before and after absorption with uninfected and HIV-1 6920-infected U937 cells.

Close modal

Fecal IgA purified from all six mice in group 1 were shown to neutralize HIV-1 SF2 by 80% at 6 and 9 mo (not shown) after booster immunization (Table V). In group 2, the fecal IgA neutralization capacity reached 50% neutralization titer at 6 mo (four of six mice) and <50% at 9 mo postbooster immunization (not shown). Fecal IgA from all other groups failed to neutralize HIV-1 SF2. This neutralization capacity was also seen against primary HIV-1 isolate 6920 at 6 and 9 mo after booster immunization in all four mice in group 1.

Table V.

HIV-1 neutralization by purified fecal IgA samples of immunized micea

HIV IsolateHIV-1 SF2HIV-1 6920
50%80%50%80%
Group      
 1 IgA pool 1 12 10 
 IgA pool 2 14 <4 
 IgA pool 3 nt nt 
 2 IgA pool 1 <4 <4 <4 
 IgA pool 2 <4 <4 
 IgA pool 3 <4 <4   
 3 IgA pool 1 <4 <4 <4 <4 
 IgA pool 2 <4 <4 <4 <4 
 4 IgA pool 1 <4 <4 <4 <4 
 IgA pool 2 <4 <4 <4 <4 
Controls      
mAb2F5 Anti-gp41 >900 300 330 160 
mAbZA1 Anti-CMV <10 <10 <10 <10 
HIV IsolateHIV-1 SF2HIV-1 6920
50%80%50%80%
Group      
 1 IgA pool 1 12 10 
 IgA pool 2 14 <4 
 IgA pool 3 nt nt 
 2 IgA pool 1 <4 <4 <4 
 IgA pool 2 <4 <4 
 IgA pool 3 <4 <4   
 3 IgA pool 1 <4 <4 <4 <4 
 IgA pool 2 <4 <4 <4 <4 
 4 IgA pool 1 <4 <4 <4 <4 
 IgA pool 2 <4 <4 <4 <4 
Controls      
mAb2F5 Anti-gp41 >900 300 330 160 
mAbZA1 Anti-CMV <10 <10 <10 <10 
a

HIV-1 neutralizing IgA titers resulting in 50 or 80% reduced HIV-1 p24 Ag detection are shown with two to three pooled IgA fractions from immunized mouse groups. nt, not tested; mAb 2F5 = human anti-ELDKWAS peptide binding monoclonal Ab; mAb ZA1, murine anti-human CMV binding monoclonal Ab. Each pool of IgA contains IgA from two mice with a final IgA concentration of 22–32 μg IgA/ml.

In group 2, the fecal IgA-neutralizing capacity was detectable at 6 mo in two of four mice and was undetectable at 9 mo postbooster immunization (not shown). HIV-1 SF2-neutralizing IgA in lung lavages was detected in two of six mice in groups 1 and 2 at 12 mo postbooster immunization (data not shown). All other groups of immunized mice lacked detectable neutralizing Abs in feces and lung lavages.

In this study our aim was to design an immunogen that induces systemic and mucosal humoral responses capable of eliminating free virus particles and protect against HIV-1 infection by inducing long-lasting B and T cell memory responses. For that reason, we decided to compare HIV-1 DNA priming/protein or peptide boost with peptide immunization only, because the first approach has previously been shown to provide a good memory response and detectable humoral immunity. Peptide or protein immunizations alone have shown a wide variability of both efficient and less efficient induction of functional Abs or cell-mediated immunity (28, 31).

In this study we have used immunogens in different combinations that contain the HIV-1 envelope gp120 and the transmembrane gp41 (gp160), a few gp41 peptides representing HIV-1-neutralizing regions, and the second external loop of the CCR5 receptor. It is known that the gp120 mediates binding and entry steps in HIV-1 infection and that the gp120-CD4 coreceptor binding induces conformational changes that activate the gp41 transmembrane regions of the envelope (32). It is also believed that the fusion structure of the envelope protein after CD4 and coreceptor binding breaks down into two coils within the gp41 monomer, and that synthetic peptides against either gp41 helical coil are able to inhibit viral infectivity (33, 34). We have included the gp41 coiled-coil peptide to obtain an Ab response that might inhibit fusion and possibly be capable of recognizing several HIV-1 subtypes. We have also included the ELDKWAS peptides, because it was previously shown that mucosal IgA, but not IgG, from HIV-seropositive individuals neutralized HIV by recognizing the ELDKWAS epitope located on the gp41, thus inhibiting HIV-1 transcytosis (35), suggesting a substantial role for sIgA.

The broadest systemic and mucosal responses were induced in mice primarily immunized with rgp160/Rev and CCR5 DNA and boosted with gp41-ELDKWAS and CCR5 peptides in mono-oleate/oleate. High, long-term serum IgG and IgA titers were obtained against the neutralizing gp41 ELDKWAS epitopes from HIV-1 clades A, B, C, and D. The lower titers of serum IgG still reacted with the gp41 coiled-coil peptide, gp120 V3 clade B, and gp160/Lai 1 year after booster immunization. Mice immunized with HIV-1 peptides only developed high serum IgG titers against the gp41 peptides, as also found in the DNA-primed animals. Similarly, serum IgA titers were equal to titers obtained in DNA-primed animals against the gp41-neutralizing ELDKWAS epitopes from clades A, B, and C, but were significantly lower or undetectable 1 year after booster immunization against the gp41 ELDKWAS epitopes from clade D, gp41 coiled-coil, and gp120 V3 peptides.

Serum IgG and IgA titers against HIV Ags did not differ significantly among the three groups of mice receiving HIV-1 Ags with or without inclusion of the CCR5 coreceptor Ag. Serum IgG and IgA responses against the CCR5-representing peptide were seen in the three mouse groups immunized with CCR5 Ag, with a significantly higher serum IgG against CCR5 in the two mouse groups receiving CCR5 Ags (p < 0.01 and p < 0.02) 6 mo after the peptide booster. This difference was lost at 12 mo of follow-up.

Differences in the long-term mucosal IgA responses in the different mouse groups were seen against HIV-1 Ags, and both HIV-specific gp41 peptides and gp160 were found in lungs and vaginal secretions. Mice immunized with HIV envelope and CCR5 DNA developed significantly higher HIV-specific IgA titers to gp41 peptides representing HIV-1 clades A–C in lungs, vaginal mucosa, and feces, whereas IgA Abs in the small intestine were directed against clades A and C and against the gp41 coiled-coil peptide. A significant difference in fecal, but not vaginal or serum, IgA reactivity against clade B gp41 peptide was found between mice given HIV-1 DNA and gp41 peptide compared with mice receiving HIV-gp41 peptides only. In mice first immunized intranasally with HIV-1 gp41/CCR5 peptides and then boosted with HIV-1/CCR5 DNA, a poor functional Ab response was obtained. These data suggest that priming the immune system with an HIV envelope immunogen in the form of a DNA plasmid better supports the development of functional virus-neutralizing Abs than intranasal priming performed with HIV-gp41 peptides even if the booster is given as a DNA plasmid. Nasal immunization of mice with HIV-1 gp160/Rev CCR5 DNA (group 1) induced higher HIV-1-specific IgA Ab titers in vaginal secretions during 6–12 mo of follow-up than in the other groups (groups 2–5), even if the numbers of responders were not significantly different in groups 1–3.

Different routes and immunogens are being used to deliver DNA to induce HIV-1-specific immune responses. It has been shown that to induce a stronger CTL response, immunizations may be given i.m. with DNA encoding CTL epitopes, followed by a gene gun booster of modified vaccinia Ankara, and that specific HIV-1 immune responses are increased as a result of the boosting strategy (13).

Intrarectal immunization has been shown to induce CTL memory in Peyer’s patches, lamina propia, and spleen in mice (36), and in macaques, CTL responses have also been found in mesenteric lymph nodes and PBMC (37); that protection could be enhanced by the use of IL-2 (38). Combinations of IL-1, IL-12, IL-18, and/or GM-CSF have been shown to be effective in inducing systemic and mucosal CTL-specific responses after nasal immunization (39). However, none of these studies reported the presence of mucosa HIV-1-IgA-specific Abs or how long these responses could be maintained, as we show in this report where mucosa Abs were enhanced by the peptide protein booster in combination with a mono-oleate fatty acid adjuvant (L3; data not shown). Our aim was to induce IgA Abs and Th cell memory; to achieve this, we believe the best way is to use a nasal immunization protocol to target mucosal B cells responsible for the generation of sIgA organized in the MALT.

Indeed, in our study, 12 mo after the last immunization, vaginal anti-gp41-specific IgA was still detectable, mainly in mice immunized with gp160/CCR5 DNA. There seemed to be a trend toward an enhanced anti-HIV Ag-specific mucosal humoral immunity in this same group of mice. The group differs in its immunization schedule by the inclusion of the CCR5 DNA and CCR5 peptide pooled with HIV-1 DNA and gp41 peptides. The use of DNA plasmid may have resulted in a higher amount of activated systemic and local lymphocytes, resulting in a longer and better preserved immune memory, as the long-term humoral mucosal immunity indicates. HIV-1-neutralizing serum Abs were highly efficient in neutralizing HIV-1 SF2 and the primary HIV-1 isolate in both plasmid DNA-primed mouse groups where the HIV-1-neutralizing capacity could be removed by adsorbing the serum with HIV-1-infected U937 cells, but only partially when uninfected cells were used. These results may suggest that the main functional Abs were directed against HIV-1-neutralizing epitopes. However, it is important to remember that the way the neutralization assay was designed was not optimal for studying the anti-CCR5-blocking capacity of the serum. Despite this, in the group 4 the serum neutralizing capacity reached 60–70% against the primary HIV-1 isolate used. This could indicate that the CCR5 coreceptor may be present at the virion envelope, as previously suggested, or that anti-idiotypic Abs for the CCR5 peptide may have induced V3-neutralizing Abs. HIV-1-neutralizing IgA purified from feces and in the lungs was detected only in mice intranasally immunized with HIV DNA with and without CCR5 plasmid.

It could be argued that the human CCR5 Ag in this mouse study would act as any foreign Ag, because the human and murine CCR5 proteins differ by ∼20% (40). It has been previously shown that anti-human or macaque CCR5-specific serum and mucosal IgG and IgA inhibit HIV-1 entry in vitro (41, 42). Serum HIV-specific IgA against the CCR5 coreceptor has been shown to develop in humans exposed to HIV as well as in individuals with the 32-bp CCR5 deletion (23). In preliminary studies we have seen how long serum IgG and IgA could last in macaques immunized with the human CCR5 as plasmid DNA and CCR5 peptide booster. Although only two monkeys were studied, both animals developed IgG and IgA Abs toward CCR5 N and second variable loop regions of the HIV-1 outer envelope protein gp120, whereas long-term serum Ab responses were seen in one of the animals (data not shown). In macaques, Abs to CCR5 remain highly stable and clearly detectable toward both the N-terminal and the second CCR5 loop region.

Previous studies have shown that plasmid HIV-1 DNA prime protein booster immunizations have resulted in long-term HIV-1-specific immunities, showing that it would be possible to induce long-term immunity to both HIV Ags and the CCR5 coreceptor without causing side effects (42). In addition, prime immunization with herpes simplex gB DNA and systemic boosting with rvacgB have been shown to be effective in inducing mucosal IgA responses in mucosally immunized mice, and these responses were increased when animals received the rvacgB as prime, followed by a booster of gB DNA (12). It is important to note that when we primed with synthetic gp41-CCR5 HIV-1 peptides and boosted with DNA/gp160-CCR5, the HIV-specific IgG or IgA Abs detected were nonneutralizing, and IgA-neutralizing Abs in the mucosa have been seen when mice were systemically primed with DNA and boosted with peptides. The reason for this is not clear, but it resembles many of the previous studies in which HIV-1 peptide immunizations have failed to induce neutralizing Abs. It is likely that the peptide-induced humoral immunity does not recognize the neutralizing epitopes presented on HIV virions. The systemic and mucosal immunity was still detectable 1 year after the booster immunization, which represents at least one-third of the life span of the mouse.

A number of vaccine studies have focused on gp120 envelope protein, because the variable regions (V1–V3) in the rgp120 envelope glycoprotein are the major targets for neutralizing Abs (28, 43, 44). However, the use of rgp120 to date has been less successful in inducing broad clade-recognizing Abs that neutralize HIV-1 primary isolates. Therefore, our immunization strategy included relatively conserved gp41 envelope components and one of the main HIV-1 coreceptors, to which some epitopes probably are exposed as cryptic and highly conserved determinants (45).

The two chosen gp41 epitopes included in our present immunization strategy thus show a high or relatively high degree of conservation, as shown in a study by Dong et al. (46). They compared 862 HIV-1 strains and showed that 90–97% of the isolates contain the LQAR sequence in the coiled-coil region, whereas the LELDKWAS region showed a more variable mix of 50–97% conserved amino acids (with amino acids L-LD-W being >97% conserved). This combination of epitopes included in a vaccine may provide the conserved epitopes that have been so difficult to find in other regions of the HIV-1 envelope even though highly conserved cross-neutralization Ab-inducing epitopes have been described in the gp120 V3 region aa GPGR (47, 48, 49) and within the CD4-binding region of gp120 (50). Additional evidence suggesting the importance of inducing Abs against these gp41 epitopes is the low frequency of Ab reactivities against these regions in HIV-infected individuals (51). In the mice used in our study the gp41 peptides are poor MHC class I binders; thus, CTL and IFN-γ responses were low or undetectable, whereas in the DNA-immunized mice, gp120 V3-specific IFN-γ was low, but detectable. The gp41 peptides were able to stimulate T cell proliferative and IL-4 responses, suggesting that a Th2-type Th cell memory may have been evoked (not shown). With the inclusion of the CCR5 DNA in our vaccine formulation, this combination has every crucial constituent involved in the fusion HIV-1 process.

The results reached by our intranasal administration of the immunogens are consistent with previous studies that showed that i.p. immunization followed by intranasal or intragastric boosts with gp41 induced mucosal and systemic Abs recognizing HIV-1 primary isolates (31). Previous studies have shown that oral or intranasal administration of PLG-encapsulated or water-dissolved plasmid DNA encoding gp160/Rev also induced Env-specific serum Abs, and that an increased level of IgA directed to gp160 was detected in the feces of immunized mice (52).

The finding that some of the HEPS individuals had seroconverted shortly after giving up sex work, have brought to light the importance of B and T cell memory to achieve protection (53). The seroconversion in this small group of previously resistant sex workers was associated with a reduction in CTL, indicating that the presentation of Ag even in small doses is important to induce a long-term immunological response. Because memory B cells can persist after immunization with low Ab production (55, 56), we measured the presence of IgA and IgG B cell synthesis in vivo in spleen and lymph nodes 1 year after intranasal immunization. We could see that the memory B cells against HIV-1 peptide and protein Ags persisted up to 1 year after booster immunization in both DNA-primed and HIV gp41 peptide-boosted mice, whereas the CCR5 peptide-immunized mice had a lower frequency of long-term B cell memory toward CCR5. Memory B cells play an important role in controlling and preventing infection. They proliferate immediately in the presence of Ags and differentiate into plasma B cells that will produce specific Abs. B cells can also present Ag, stimulating T cell and cytokine responses (57, 58, 59). To evaluate the specific immune responses induced by our immunogens, we performed a number of immunizations with rgp160/Rev DNA/ELDKWAS peptide, with and without the CCR5 peptide. No responses detected against the CCR5 coreceptor were seen in animals without the CCR5 Ag, whereas specific B cell responses were detected against rgp160; gp41 clades A, B, C, and D; gp41 coiled-coil peptide; and whole recombinant gp41 when CR5 was included.

Previous investigations have shown that Abs play a crucial role in prevention against infection. Antiviral and protective activities were detected when neutralizing Abs were i.v. administered passively (14, 49, 59, 60, 61). These data clearly suggest that systemic Abs can provide protection against mucosal virus exposure, but because IgG or monomeric IgA in serum and plasma will not be actively transported across mucosa, whereas secretory IgA or IgM will, we believe that mucosal IgA Abs could play an important role and also inhibit epithelial HIV transcytosis (21). Probably the most robust immunity against a virus that causes chronic infection such as HIV would be to have a systemic and mucosal B cell repertoire capable of neutralizing the virus, and a systemic as well as local T cell response, with both Th and cytotoxic properties, should be able to prevent entry of the virus. In most cases where the inhibition of virus entry via mucosa fails, a systemic HIV-neutralizing B and T cell response may still be preventive, but we believe that this level of immunity, when lacking the immunity in the mucosal compartments, may have a smaller chance of being efficient over the long term. Our study suggests the intranasal route for administering HIV-1 DNA plasmid vaccines combined with relevant synthetic HIV-1 peptides as a non-live, safe, and promising vaccine combination with the capacity to provide potent functional antiviral humoral immunity.

In conclusion, HIV-1 gp41-specific IgA were found in feces and lung and vaginal secretions up to 12 mo after immunization. HIV-specific IgG and IgA Abs and memory B cells directed against putative neutralizing epitopes were detectable in spleen and draining lymph nodes against gp41/MN; gp41 clades A, B, and C; rgp160; and CCR5 loop peptide. This response was induced by a vaccine that includes critical fusion-dependent determinants. Because memory B cells accumulate with every immunization even when Abs are not detectable in serum, we measured IgG and IgA B cell stimulation in vitro in spleen and lymph nodes that reflected the presence of long-term immunity. Although innumerable vaccine studies promoted the idea of boosting cellular immunity, the induction of long-lasting memory B cells is still an unresolved task in the development of a preventive HIV vaccine; our mucosal approach could provide some clue of how this may be accomplished.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants from the Swedish Research Council, the Swedish Medical Society, KI Fonder, and Swedish Physicians against AIDS.

3

Abbreviations used in this paper: gB, gp of herpes simplex virus; HEPS, highly exposed, persistently seronegative; rvacgB, gB expressed and attenuated recombinant vaccinia virus vector; sIgA, secretory IgA; SHIV, simian-human immunodeficiency virus; NSI, non-syncytium-inducing.

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