Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120

Recombinant gp120 of the HIV-1(IIIB) isolate (BH10 clone) has been mutated to form the PR12 protein with the first 74 C-terminal amino acids and the V1, V2 and V3 hypervariable loops deleted. A variety of studies have shown that the CD4 binding domain (CD4bd) is very well exposed in PR12 in contrast to rgp120(LAI). Using PR12 for selection of human monoclonal antibodies (MAbs) from HIV-infected individuals, five MAbs were generated with specificities to the epitopes overlapping the CD4bd (1570A,1570C,1570D,1595 and 1599). The three MAbs, 1570A, C and D, generated from one HIV-infected individual, represent one MAb as determined by sequence analysis of the V(H)3 region. Since the epitopes overlapping the CD4bd exhibit variability among HIV-1 clades, the specificity of anti-CD4bd MAbs were distinguished by differing patterns of binding to recombinant envelope proteins derived from clade A, B, C, D and E viruses. The PR12-selected MAbs were also compared with a panel of gp120-selected anti-CD4bd MAbs and showed a different range of specificities. MAb 1599 is clade B specific, MAb 1595 reacts with the A, B and D clades, while MAb 1570 recognises the most conserved epitope, as it binds to all proteins. The results show that the exposure of different epitopes in the CD4bd of the PR12 protein allows this protein to serve as an immunogen and to induce anti-CD4bd antibodies.     

Focusing the immune response on the V3 loop, a neutralizing epitope of the HIV-1 gp120 envelope

Rabbits were immunized with a novel regimen designed to focus the immune response on a single neutralizing epitope of HIV-1 gp120 and thereby preferentially induce neutralizing antibodies (Abs). Animals were primed with gp120 DNA from a clade A Env bearing the GPGR V3 motif and/or a clade C Env bearing the GPGQ V3 motif, and boosted with one or more fusion proteins containing V3 sequences from clades A, B and/or C. Immune sera neutralized three of four Tier 1 primary isolates, including strains heterologous to the immunizing strains, and potent cross-clade-neutralizing activity was demonstrated against V3 chimeric pseudoviruses carrying in a Tier 1 Env, the consensus V3 sequences from clades A1, AG, B, AE, or F. The broadest and most potent neutralizing responses were elicited with the clade C gp120 DNA and a combination of V3-fusion proteins from clades A, B and C. Neutralizing activity was primarily due to V3-specific Abs. The results demonstrate that the immune response can be focused on a neutralizing epitope and show that the anti-V3 Abs induced recognize a diverse set of V3 loops.     

High titer HIV-1 V3-specific antibodies with broad reactivity but low neutralizing potency in acute infection and following vaccination

Identifying the earliest neutralizing antibody specificities that are elicited following infection or vaccination by HIV-1 is an important objective of current HIV/AIDS vaccine research. We have shown previously that transplantation of HIV-1 V3 epitopes into an HIV-2 envelope (Env) scaffold provides a sensitive and specific means to detect and quantify HIV-1 V3 epitope specific neutralizing antibodies (Nabs) in human sera. Here, we employ this HIV-2/HIV-1 V3 scaffolding strategy to study the kinetics of development and breadth of V3-specific Nabs in longitudinal sera from individuals acutely infected with clade C or clade B HIV-1 and in human subjects immunized with clade B HIV-1 immunogens. HIV-2/HIV-1 chimeras containing V3 sequences matched to virus type (HIV-2 or HIV-1), subtype (clade B or C), or strain (autologous or heterologous) were used as test reagents. We found that by 3-8 weeks post infection, 12 of 14 clade C subjects had a median IC₅₀ V3-specific Nab titer of 1:700 against chimeric viruses containing a heterologous clade C V3. By 5 months post-infection, all 14 subjects were positive for V3-specific Nabs with median titers of 1:8000 against heterologous clade C V3 and 1:1300 against clade B V3. Two acutely infected clade B patients developed heterologous clade B V3-specific Nabs at titers of 1:300 and 1:1800 by 13 weeks of infection and 1:5000 and 1:11000 by 7 months of infection. Titers were not different against chimeras containing autologous clade B V3 sequences. Each of 10 uninfected normal human volunteers who were immunized with clade B HIV-1 Env immunogens, but none of five sham immunized control subjects, developed V3-specific Nabs titers as high as 1:3000 (median 1:1300; range 1:700-1:3000). None of the HIV-1 infected or vaccinated subjects had antibodies that neutralized primary HIV-1 virus strains. These results indicate that high-titer, broadly reactive V3-specific antibodies are among the first to be elicited during acute and early HIV-1 infection and following vaccination but these antibodies lack neutralizing potency against primary HIV-1 viruses, which effectively shield V3 from antibody binding to the functional Env trimer.     

Standardized assessment of NAb responses elicited in rhesus monkeys immunized with single- or multi-clade HIV-1 envelope immunogens

The genetic diversity of HIV-1 envelope glycoproteins (Env) remains a major obstacle to the development of an antibody-based AIDS vaccine. The present studies examine the breadth and magnitude of neutralizing antibody (NAb) responses in rhesus monkeys after immunization with DNA prime-recombinant adenovirus (rAd) boost vaccines encoding either single or multiple genetically distant Env immunogens, and subsequently challenged with a pathogenic simian-human immunodeficiency virus (SHIV-89.6P). Using a standardized multi-tier panel of reference Env pseudoviruses for NAb assessment, we show that monkeys immunized with a mixture of Env immunogens (clades A, B, and C) exhibited a greater breadth of NAb activity against neutralization-sensitive Tier 1 viruses following both vaccination and challenge compared to monkeys immunized with a single Env immunogen (clade B or C). However, all groups of Env-vaccinated monkeys demonstrated only limited neutralizing activity against Tier 2 pseudoviruses, which are more characteristic of the neutralization sensitivity of circulating HIV-1. Notably, the development of a post-challenge NAb response against SHIV-89.6P was similar in monkeys receiving either clade B, clade C, or clade A+B+C Env immunogens, suggesting cross-clade priming of NAb responses. In addition, vaccines encoding Env immunogens heterologous to SHIV-89.6P primed for a rapid anamnestic NAb response following infection compared to vaccines lacking an Env component. These results show that DNA/rAd immunization with multiple diverse Env immunogens is a viable approach for enhancing the breadth of NAb responses against HIV-1, and suggest that Env immunogens can prime for anamnestic NAb responses against a heterologous challenge virus.     

Isolation and characterization of monoclonal antibodies elicited by trimeric HIV-1 Env gp140 protein immunogens

Eleven anti-HIV Env monoclonal antibodies (MAbs) were isolated from mice immunized with soluble Env proteins derived from the clade B Env, SF162, or DeltaV2 (a derivative of SF162 lacking the V2 loop). All six anti-gp120 MAbs studied, neutralized SF162 and their activities were dependent by the glycosylation patterns of the V1, V2 or V3 loops. Only one anti-gp120 MAb (an anti-V3 MAb) displayed cross-neutralizing activity, which was influenced by the type of V1 loop present on the target heterologous viruses. None of the five anti-gp41 MAbs studied displayed anti-SF162 neutralizing activity. Our studies indicate that the current limitation of soluble HIV Env gp140 immunogens to elicit robust cross-reactive neutralizing antibody responses is not only due to the elicitation of high titers of homologous antibodies but also due to the elicitation of antibodies whose epitopes are naturally occluded, or not present, on the virion-associated Env.     

Modulation of polymorphonuclear cell interleukin-8 secretion by human monoclonal antibodies to type 8 pneumococcal capsular polysaccharide

Pneumococcal capsular polysaccharide (PS) vaccines induce type-specific immunoglobulin M (IgM), IgG, and IgA. Type-specific IgG to the PS is sufficient to confer protection against the homologous serotype of the pneumococcus, but the efficacies of type-specific IgM and IgA are less well understood. We examined the in vitro activities and efficacies in mice of two human monoclonal antibodies (MAbs) to type 8 PS, NAD (IgA) and D11 (IgM). MAb-mediated opsonophagocytic killing was evaluated after coculture of type 8 pneumococci with human polymorphonuclear cells (PMNs), type-specific or control MAbs, and human complement sources. The effects of the MAbs on PMN interleukin-8 (IL-8) and IL-6 secretion were determined in supernatants from cocultures containing pneumococci and PMNs by enzyme-linked immunosorbent assay. MAb efficacy was determined in an intratracheal model of type 8 infection in mice with classical complement pathway deficiency. Both MAbs were protective in 100% of infected mice. Neither MAb promoted a significant amount of killing of type 8 pneumococci compared to its isotype control MAb. Both type-specific MAbs mediated complement-dependent modulation of PMN IL-8 secretion, with increased secretion at effector/target (E:T) ratios of 500:1 and 50:1 and reduced secretion at 1:5. Trypan blue staining revealed that PMNs cocultured with D11 were less viable at an E:T ratio of 1:5 than PMNs cocultured with the control MAb. PMN IL-6 secretion was increased by both type-specific and control MAbs. These results suggest that certain type-specific IgM and IgAs might contribute to host defense by modulation of the inflammatory response to pneumococci.     

Polyvalent HIV-1 Env vaccine formulations delivered by the DNA priming plus protein boosting approach are effective in generating neutralizing antibodies against primary human immunodeficiency virus type 1 isolates from subtypes A, B, C, D and E

A major challenge in developing an HIV-1 vaccine is to identify immunogens and their delivery methods that can elicit broad neutralizing antibodies against primary isolates of different genetic subtypes. Recently, we demonstrated that priming with DNA vaccines expressing primary HIV-1 envelope glycoprotein (Env) followed by recombinant Env protein boosting was successful in generating positive neutralizing antibody responses against a clade B primary HIV-1 isolate, JR-FL, that was not easily neutralized. In the current study, we examined whether the DNA priming plus recombinant protein boosting approach delivering a polyvalent primary Env formulation was able to generate neutralizing antibodies against primary HIV-1 viral isolates from various genetic subtypes. New Zealand White rabbits were first immunized with DNA vaccines expressing one, three or eight primary HIV-1 gp120 antigens delivered by a gene gun followed by recombinant gp120 protein boosting. Neutralizing antibody responses were examined by two independently executed neutralization assays: the first one was a single round infection neutralization assay against a panel of 10 primary HIV-1 isolates of subtypes A, B, C and E and the second one used the PhenoSense assay against a panel of 12 pseudovirues expressing primary HIV-1 Env antigens from subtypes A, B, C, D and E as well as 2 pseudoviruses expressing the Env antigens from MN and NL4-3 viruses. Rabbit sera immunized with the DNA priming plus protein boosting approach, but not DNA vaccine alone or Env protein alone, were capable of neutralizing 7 of 10 viruses in the first assay and 12 of 14 viruses in the second assay. More importantly, sera immunized with the polyvalent Env antigens were able to neutralize a significantly higher percentage of viruses than the sera immunized with the monovalent antigens. Our results suggest that DNA priming followed by recombinant Env protein boosting can be used to deliver polyvalent Env-antigen-based HIV-1 vaccines to elicit neutralizing antibody responses against viruses with diverse genetic sequence variations.     

Correlation between In Vitro Complement Deposition and Passive Mouse Protection of Anti-Pneumococcal Surface Protein A Monoclonal Antibodies

The shortcomings of the licensed polysaccharide-based pneumococcal vaccine are driving efforts toward development of a protein-based vaccine that is serotype independent and effective in all age groups. An opsonophagocytic killing assay (OPKA) is used to evaluate the antibody response against polysaccharide-based pneumococcal vaccines. However, the OPKA is not reliable for noncapsular antigens. Thus, there is a need to develop an in vitro surrogate for protection for protein vaccine candidates like pneumococcal surface antigen A (PspA). PspA is a serologically variable cell surface virulence factor. Based on its sequence, PspA has been classified into families 1 (clade 1 and 2), 2 (clades 3, 4 and 5), and 3 (clade 6). Here, we report the characterization of 18 IgG anti-PspA monoclonal antibodies (anti-PspA^hkR36A MAbs) generated from mice immunized with heat-killed strain R36A (clade 2). An enzyme-linked immunosorbent assay (ELISA)-based analysis of the reactivity of the MAbs with recombinant PspAs from the 6 clades indicated that they were family 1 specific. This was confirmed by flow cytometry using a hyperimmune serum generated against PspA from R36A. Eight MAbs that bind at least one clade 1- and clade 2-expressing strain were evaluated for complement deposition, bactericidal activity, and passive protection. The anti-PspA^hkR36A MAb-dependent deposition of complement on pneumococci showed a positive correlation with passive protection against strain WU2 (r = 0.8783, P = 0.0041). All of our protective MAbs showed bactericidal activity; however, not all MAbs that exhibited bactericidal activity conferred protection in vivo. The protective MAbs described here can be used to identify conserved protection eliciting B cell epitopes for engineering a superior PspA-based vaccine.     

Neutralization of HIV-1 primary isolate by ELDKWA-specific murine monoclonal antibodies

ELDKWA on HIV-1 gp41 is a conserved epitope recognized by one broadly neutralizing monoclonal antibody 2F5, which is a promising candidate target for vaccine design. Here we report two ELDKWA-specific monoclonal antibodies (mAbs), 18F11 and 7E10, that were screened from the splenocytes of mice immunized by recombinant GST-(ELDKWA)4 protein. In further evaluation, these mAbs exhibited appreciable neutralizing activities against HIV-1 primary isolate 92US675 (clade B) with IC50 (50% inhibition concentration) of 6.84 +/- 0.36 microg/ml and 10.66 +/- 1.69 microg/ml, respectively. Unexpectedly, neither of these two murine mAbs could neutralize laboratory-adapted strain HIV-1 IIIB (clade B). As a control, human mAb 2F5 neutralized both primary and laboratory-adapted strains. These data strongly suggest that ELDKWA-specific antibodies induced by different antigenic formats show different neutralizing activities against HIV-1, which implies another complication in the development of effective vaccines.     

Immunization of rhesus macaques with a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine elicits protective antibody response against simian human immunodeficiency virus of R5 phenotype

The immunogenicity of a poylvalent HIV-1 vaccine comprised of Env antigens from primary R5 isolates was evaluated in rhesus macaques. DNA vaccines encoding four Env antigens from multiple HIV-1 subtypes and HIV-1 Gag antigen from a single subtype elicited a persistent level of binding antibodies to gp120 from multiple HIV-1 isolates that were markedly enhanced following boosting with homologous gp120 proteins in QS-21 adjuvant irrespective of the route of DNA immunization. These sera neutralized homologous and, to a lesser degree, heterologous HIV-1 isolates. Four of the six immunized animals were completely protected following rectal challenge with a SHIV encoding Env from HIV-1(Ba-L), whereas the virus load was reduced in the remaining animals compared to na?ve controls. Hence priming with DNA encoding Env antigens from multiple HIV-1 clades followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of protecting macaques against mucosal transmission of R5 tropic SHIV isolate.     

Human monoclonal antibodies reactive with cell surface antigens on human leukemia cell lines: many antibodies are (auto)antibodies

Primary Epstein Barr Virus (EBV) transformants from peripheral blood mononuclear cells (PBM) established in macrocultures were screened for the secretion of antibodies reactive with cell surface antigens on one or another of two indicator human leukemic cell lines and fused with the HMMA2.11TG/O human fusion partner. Human monoclonal antibodies (HuMAbs) were readily obtained. Relative oligoclonality of the primary EBV macrocultures was documented by the number of antibody secreting hybridomas (1-100%). The method permitted preselection for fusion of transformants producing antibodies of certain specificities and/or class. Fourteen HuMAbs, primarily of the IgM class, have been obtained. Those IgM HuMAbs obtained from patients with active diseases, e.g. Acute Lymphoblastic Leukemia (3 HuMAbs), and HIV infection (4 HuMAbs), were found to have a relatively broad spectrum of reactivity with cell lines of various hematopoietic lineages and normal cells, although several show selective reactivity with T cell lineage tumors or a selected population of cells. HuMAbs from normal donors of both the IgG and IgM class were obtained. The IgM HuMAbs from one volunteer reacted primarily with autologous and allogeneic macrophages (autologous PBM from the other patients were not available) as well as a diverse number of hematopoietic cell lines. From others, the IgG HuMAbs demonstrated a more restricted spectrum of reactivity, while the IgM HuMAbs reacted with both autologous and allogeneic normal cells. Thus, the B cell repertoire contains cells capable of secreting cell surface reactive antibodies and many of these antibodies express characteristics of autoantibodies. Those that did not react with autologous or allogeneic PBM may react with other autoantigens which have been expressed on the malignant cells used as screening targets or may represent true antitumor antibodies.     

Molecular analysis of neutralizing epitopes on outer surface proteins A and B of Borrelia burgdorferi.

The neutralizing epitopes of the major outer surface proteins A and B (OspA and OspB) of Borrelia burgdorferi B31 were investigated by epitope mapping using overlapping synthetic peptides, encompassing full-length OspA and OspB, and antiborrelial monoclonal antibodies (MAbs). OspA MAb N4B12 and OspB MAbs N5G5, W7C2, and P4D1 displayed a complement-independent antiborrelial activity, and complement failed to enhance the antiborrelial activity, as measured by a sensitive colorimetric assay. A combination of N4B12 with N5G5 displayed a higher antiborrelial activity than did the MAbs individually. OspA MAbs B3G11 and L3B5, however, exhibited a significant antiborrelial activity only in the presence of complement. Epitope mapping showed that B3G11 bound to one OspA synthetic peptide with the sequence of amino acids 247 to 256 (QYDSNGTKLE) and produced more than sixfold-higher reactivity than with other sequences, as measured by an enzyme-linked immunosorbent assay. OspB MAb N5G5 bound to an OspB peptide with the sequence of amino acids 211 to 220 (TLKREIEKDG), yielding at least threefold-higher reactivity than with other sequences. These two peptide sequences were found to contain neutralizing epitopes. Other MAbs had weak binding activities with the synthetic peptides, and their specific epitopes remain to be further analyzed. Thus, this study demonstrated both complement-independent and complement-dependent antiborrelial MAbs and identified the linear epitopes on OspA and OspB capable of inducing neutralizing antibody responses.     

Monoclonal and polyclonal antibodies against human ferritin, a nonspecific tumor marker

The aim of this study was to produce monoclonal and polyclonal antibodies against a nonspecific tumor marker, human ferritin. Hyperimmune ICR mice produced polyclonal antibodies after injection with 0.5 mL pristane, and were injected with NS-1 myeloma cells 2 weeks later. Hyperimmune Balb/c mice were used for the production of monoclonal antibodies (MAbs). Mice were immunized four times, given a final boost, and their spleen cells were collected and fused with NS-1 myeloma cells under the presence of PEG 1500. The fused cells were then selected in the HAT-RPMIX medium. Anti-ferritin antibody-secreting hybridoma cell lines with high titer were cloned by enzyme-linked immunoadsorbent assay (ELISA) and then subcloned by limiting dilution in 15% fetal bovine serum (FBS) HT-RPMIX medium. Five murine hybridoma-producing antiferritin MAbs were obtained and designated 1AD11F9, 1AD11E11, 2AD11D2, 2AD11A5, and 3AD11G8. Isotypes of these MAbs were identified as IgM heavy chain and kappa light chain. Hitrap Protein A and Hitrap IgM purification column were used for the purification of polyclonal and monoclonal antibodies, respectively.     

Cross-reactive monoclonal antibodies to multiple HIV-1 subtype and SIVcpz envelope glycoproteins

The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE, and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross-reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers.     

Monoclonal antibodies against Candida tropicalis mannan: antigen detection by enzyme immunoassay and immunofluorescence.

Three strains of mice were immunized with Candida tropicalis cell walls, and antibodies against mannan were detected by indirect enzyme immunoassay (EIA) in 3 of 9 BALB/c mice, 4 of 11 C57BL/6 mice, and 4 of 8 CFW mice. Responding mice produced immunoglobulin M (IgM), but IgG was not detected in their sera. Fusion of the high-responder BALB/c mouse with a plasmacytoma cell line resulted in 41 clones secreting antimannan monoclonal antibodies (MAbs). Four clones selected for propagation included one IgM and one IgG MAb that reacted with mannans of Candida albicans serotypes A and B and of C. tropicalis and two IgM MAbs specific for an epitope only in the mannans of C. albicans serotype A and C. tropicalis. One of the IgM MAbs, CB6, was an effective substitute for rabbit antibodies in the double-antibody sandwich EIA to detect antigenemia produced in rabbits infected with C. albicans A or C. tropicalis. It could function either as the peroxidase-conjugated indicator antibody or as the capture antibody. Two MAbs, CB6 (C. tropicalis and C. albicans A specific) and AC3 (C. tropicalis and C. albicans A and B specific), functioned in place of polyclonal antisera in the serotyping of C. albicans by immunofluorescence. There was 95.8% agreement in the results of serotyping using MAbs as reagents compared with rabbit antisera. Competitive inhibition in EIA between CB6 and monospecific antisera against C. albicans factors 1, 4, and 6 indicated that CB6 binds to an epitope which is probably factor 6. Serologic similarity between factor 4 and the binding site of MAb AC3 was also determined.     

Production and characterization of monoclonal antibodies reactive with the mycelial and yeast phases of Penicillium marneffei.

A definitive tissue diagnosis of Penicillium marneffei is hampered by a microscopic similarity to the yeast form of Histoplasma capsulatum and Cryptococcus neoformans. In order to obtain a better discrimination for accurate diagnosis, monoclonal antibodies (MAbs) were produced from hybridomas raised from Balb/c mice immunized with mycelial culture filtrate. By indirect immunofluorescent or immunoblot analysis, one immunoglobulin (Ig) G1 (3C2) MAb and three IgM (8B11, 3B9 and 8C3) MAbs were found to react strongly with P. marneffei antigens. In the immunoblots, the MAbs 8B11 and 3B9 reacted most strongly with a high molecular weight component (> 200 kDa) produced during either the mycelial or yeast phase of fungal growth. The immunoreactive epitopes for these two IgM MAbs were most likely associated with carbohydrate moieties, judging from their susceptibility to periodate oxidation and concanavalin A binding. This is in contrast to the immunoreactive epitopes for the MAbs 8C3 and 3C2, which were resistant to destruction by periodate treatment and did not bind to the lectin. Judging from immunofluorescent intensity, the three IgM MAbs could react strongly with the yeast cells present in the tissue biopsies of patients with P. marneffei infection.     

Human monoclonal antibodies directed against toxins A and B prevent Clostridium difficile-induced mortality in hamsters

Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, and recent outbreaks of strains with increased virulence underscore the importance of identifying novel approaches to treat and prevent relapse of Clostridium difficile-associated diarrhea (CDAD). CDAD pathology is induced by two exotoxins, toxin A and toxin B, which have been shown to be cytotoxic and, in the case of toxin A, enterotoxic. In this report we describe fully human monoclonal antibodies (HuMAbs) that neutralize these toxins and prevent disease in hamsters. Transgenic mice carrying human immunoglobulin genes were used to isolate HuMAbs that neutralize the cytotoxic effects of either toxin A or toxin B in cell-based in vitro neutralization assays. Three anti-toxin A HuMAbs (3H2, CDA1, and 1B11) could all inhibit the enterotoxicity of toxin A in mouse intestinal loops and the in vivo toxicity in a systemic mouse model. Four anti-toxin B HuMAbs (MDX-1388, 103-174, 1G10, and 2A11) could neutralize cytotoxicity in vitro, although systemic toxicity in the mouse could not be neutralized. Anti-toxin A HuMAb CDA1 and anti-toxin B HuMAb MDX-1388 were tested in the well-established hamster model of C. difficile disease. CDA1 alone resulted in a statistically significant reduction of mortality in hamsters; however, the combination treatment offered enhanced protection. Compared to controls, combination therapy reduced mortality from 100% to 45% (P<0.0001) in the primary disease hamster model and from 78% to 32% (P<0.0001) in the less stringent relapse model.     

Structure-function relationships for human antibodies to pneumococcal capsular polysaccharide from transgenic mice with human immunoglobulin Loci

To investigate the influence of antibody structure and specificity on antibody efficacy against Streptococcus pneumoniae, human monospecific antibodies (MAbs) to serotype 3 pneumococcal capsular polysaccharide (PPS-3) were generated from transgenic mice reconstituted with human immunoglobulin loci (XenoMouse mice) vaccinated with a PPS-3-tetanus toxoid conjugate and their molecular genetic structures, epitope specificities, and protective efficacies in normal and complement-deficient mice were determined. Nucleic acid sequence analysis of three MAbs (A7, 1A2, and 7C5) revealed that they use two different V(H)3 genes (A7 and 1A2 both use V3-15) and three different V(kappa) gene segments. The MAbs were found to have similar affinities for PPS-3 but different epitope specificities and CDR3 regions. Both A7 and 7C5 had a lysine at the V(H)-D junction, whereas 1A2 had a threonine. Challenge experiments with serotype 3 S. pneumoniae in BALB/c mice revealed that both 10- and 1- micro g doses of A7 and 7C5 were protective, while only a 10- micro g dose of 1A2 was protective. Both A7 and 7C5 were also protective in mice lacking either an intact alternative (FB(-/-)) or classical (C4(-/-)) complement pathway, but 1A2 was not protective in either strain. Our data suggest that PPS-3 consists of epitopes that can elicit both highly protective and less protective antibodies and that the superior efficacies of certain antibodies may be a function of their structures and/or specificities. Further investigation of relationships between structure, specificity, and efficacy for defined MAbs to PPS may identify antibody features that might be useful surrogates for antibody (and vaccine) efficacy.     

Anti-P30-52 monoclonal antibody cross-reacted to Env V3 and inhibited the viral multiplication of HIV-1-infected MT-4 cells.

It is well known that the anti-p17 antibody titer decreases with the disease progression among human immunodeficiency virus type 1 (HIV-1) carriers. We previously established several murine anti-p17 monoclonal antibodies (MAbs) to investigate the immunological role of p17, and to further characterize these MAbs, we examined the anti-p17 antibody titer in serum of a patient who was a long-term nonprogressor with hemophilia, and found that the antibody for the p17-derivative peptide from amino acid residues 30 to 52 (P30-52) cross-reacted to the third variable region of the envelope glycoprotein of HIV-1, Env V3. In the present study, we primed mice with P30-52 to establish anti-P30-52 MAbs (P30-52 MAbs), and examined their affinity and whether they suppressed the viral multiplication of HIV-1-infected MT-4 (HTLV-1-transformed CD4+ T-cell line) cells, in a TCID50 assay. At the same time, an anti-Env V3 MAb (Env V3 MAb) was also established and examined as above. The IgM-type P30-52 MAb and Env V3 MAb showed heteroclitic binding, and the IgM-type P30-52 MAb inhibited the viral multiplication. We also found that an increase of fragmented DNA of HIV-1-infected MT-4 cells co-cultured with P30-52 MAbs. Because DNA fragmentation is one of the features of programmed cell death, the viral multiplication may be suppressed by the apoptosis of HIV-1-infected MT-4 cells co-cultured with P30-52 MAbs. Though the relationship between cross-reactivity and the inhibition mechanism of multiplication of HIV-1 is unclear, P30-52 of p17 may well be a useful region of viral proteins for the development of therapeutic and vaccination strategies.     

Cross-Clade-Specific Cytotoxic T Lymphocytes in HIV-1-Infected Children

We studied cytotoxic T lymphocyte (CTL) cross-reactivity between human immunodeficiency virus type 1 (HIV-1) subtypes within a group of infants infected with either HIV-1 B or non-B clade. Fifteen children were infected with a clade B virus. Nine were infected with non-B virus, including two clade A, four clade D, two clade F, and one clade G. CTL activities fromin vitroactivated peripheral blood mononuclear cells were tested against autologous cell line infected with recombinant vaccinia viruses encoding for Env, Gag, Pol, or Nef proteins from a clade A or B isolate. HIV-1-specific CTL elicited from infection with clade B virus could lyse targets expressing clade A proteins, and vice versa. In infants with positive CTL responses, cross-clade recognition was predominant and was detected within 88% of the Pol, 83% of the Nef, 67% of the Gag, and 55% of the Env responders. Longitudinal studies showed that CTL cross-reactivity to both B and A targets was stable for several years. Elicitation of CTL reactivities capable of elimination of virus-infected cells is an important goal for the development of an efficient AIDS vaccine. The significant cross-reactivity of CTL shown in this study supports the concept that vaccines developed using a single-clade immunogen may be applicable to induce broadly reactive T cell responses.