Factors affecting the immunogenicity and potency of tetanus toxoid: implications for the elimination of neonatal and non-neonatal tetanus as public health problems

The neutralizing activity of anti-V3 monoclonal antibodies (MAbs) and anti-HIV-1 immune sera was tested against HIV-1 laboratory strains and African primary isolates. Neutralization was investigated in Phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cell (PBMC) cultures by means of two distinct viral titer reduction assays. In these assays, virus was detected by means of either p24 antigen measurement using ELISA or HIV provirus synthesis using PCR, respectively. Anti-V3 MAbs and anti-HIV-1 immune sera neutralized efficiently the homologous laboratory HIV-1 strains used for eliciting immune response but showed no neutralizing activity against most primary isolates. The two neutralization assays used provided similar results. However, a PCR-based assay circumvented the limitations due to low levels of virus replication. The mechanism of resistance of the primary isolates to neutralizing antibodies was complex and was not simply predicted by partial sequence determination of the epitopes. This points out the need for reliable neutralization assays of HIV-1 primary isolates in order to evaluate the role of humoral immunity during HIV-1 infection and for future vaccine strategies.     

Sera from HIV-1 infected individuals in all stages of disease preferentially recognize the V3 loop of the prototypic macrophage-tropic glycoprotein gp120 ADA

The outer membrane glycoprotein gp120 and the transmembrane glycoprotein gp41 are predominant targets of the humoral immune response to infection by human immunodeficiency virus type 1. The third hypervariable region (V3 loop) is the principal neutralizing domain and is the primary target of neutralizing antibodies directed against the envelope proteins of HIV-1. The V3 loop is also the major determinant for HIV-1 cell-specific tropism. To further characterize the humoral immune response directed against the gp120 envelope proteins, we expressed two prototypic gp120 envelope proteins (LAI/HXB2 and ADA) and chimeric gp120 envelope proteins in stable transfected Drosophila melanogaster Schneider 2 cells. Sera from four infected adults over the course of infection [McNearney et al. (1992) Proc. natn. Acad. Sci. U.S.A. 89, p. 10,242] were assayed for reactivity with the respective envelope proteins. Sera obtained at early stages preferentially recognized the gp120 envelope protein ADA, whereas in later stages of infection the sera showed diminished reactivity with both gp120 LAI/HXB2 and gp120 ADA. Chimeric envelope proteins revealed that the humoral response was directed primarily against the V3 loop of gp120 ADA. Furthermore, 22 sera from HIV-1 infected individuals in different stages of the disease were tested. Reactivity of sera with the gp120 envelope protein ADA was seven-fold higher than with the gp120 envelope protein LAI/HXB2. Our results suggest that the humoral immune response is preferentially elicited against the V3 loop of the prototypic macrophage-tropic gp120 envelope protein ADA.     

Potent neutralization of primary HIV-1 isolates by antibodies directed against epitopes present in the V1/V2 domain of HIV-1 gp120

Although it is known that some human immune sera possess potent neutralizing activities for primary viruses, the identity of the target epitopes mediating this neutralization is unknown, and currently available immunogens have not been able to induce such activities. Using recombinant fusion glycoproteins expressing native V1/V2 domains of gp120 we have found that sera from a subset of HIV-1-infected humans contain antibodies that recognize broadly conserved V1/V2 epitopes. Such antibodies were isolated from one human serum by affinity chromatography on a column containing a V1/V2 fusion protein, and shown to efficiently neutralize several macrophage-tropic HIV-1 isolates. Rodents immunized with the purified V1/V2 fusion protein produced antibodies reactive with unrelated V1/V2 fusion proteins and with heterologous gp120s. V1/V2-specific immunoglobulins isolated from sera of these animals by affinity chromatography also possessed potent neutralization activity for several primary HIV-1 isolates. These results indicate that the V1/V2 domain of HIV-1 gp120 contains conserved epitopes that mediate potent neutralization of primary viruses, and suggest that subunit vaccines that efficiently induce such antibodies may provide protective humoral immunity against clinically relevant HIV-1 isolates.     

The humoral immune response to human T-cell lymphotropic virus type 1 envelope glycoprotein gp46 is directed primarily against conformational epitopes

Individuals infected with human T-cell lymphotropic virus type 1 (HTLV-1) develop a robust immune response to the surface envelope glycoprotein gp46 that is partially protective. The relative contribution of antibodies to conformation-dependent epitopes, including those mediating virus neutralization as part of the humoral immune response, is not well defined. We assess in this report the relationship between defined linear and conformational epitopes and the antibodies elicited to these domains. First, five monoclonal antibodies to linear epitopes within gp46 were evaluated for their ability to abrogate binding of three human monoclonal antibodies that inhibit HTLV-1-mediated syncytia formation and recognize conformational epitopes. Binding of antibodies to conformational epitopes was unaffected by antibodies to linear epitopes throughout the carboxy-terminal half and central domain of HTLV-1 gp46. Second, an enzyme-linked immunoadsorbent assay was developed and used to measure serum antibodies to native and denatured gp46 from HTLV-1-infected individuals. In sera from infected individuals, reactivity to denatured gp46 had an average of 15% of the reactivity observed to native gp46. Third, serum antibodies from 24 of 25 of HTLV-1-infected individuals inhibited binding of a neutralizing human monoclonal antibody, PRH-7A, to a conformational epitope on gp46 that is common to HTLV-1 and -2. Thus, antibodies to conformational epitopes comprise the majority of the immune response to HTLV-1 gp46, and the epitopes recognized by these antibodies do not appear to involve sequences in previously described immunodominant linear epitopes.     

Role of E4 in eliciting CD4 T-cell and B-cell responses to adenovirus vectors delivered to murine and nonhuman primate lungs

Adenovirus vectors delivered to lung are being considered in the treatment of cystic fibrosis (CF). Vectors from which E1 has been deleted elicit T- and B-cell responses which confound their use in the treatment of chronic diseases such as CF. In this study, we directly compare the biology of an adenovirus vector from which E1 has been deleted to that of one from which E1 and E4 have been deleted, following intratracheal instillation into mouse and nonhuman primate lung. Evaluation of the E1 deletion vector in C57BL/6 mice demonstrated dose-dependent activation of both CD4 T cells (i.e., TH1 and TH2 subsets) and neutralizing antibodies to viral capsid proteins. Deletion of E4 and E1 had little impact on the CD4 T-cell proliferative response and cytolytic activity of CD8 T cells against target cells expressing viral antigens. Analysis of T-cell subsets from mice exposed to the vector from which E1 and E4 had been deleted demonstrated preservation of TH1 responses with markedly diminished TH2 responses compared to the vector with the deletion of E1. This effect was associated with reduced TH2-dependent immunoglobulin isotypes and markedly diminished neutralizing antibodies. Similar results were obtained in nonhuman primates. These studies indicate that the vector genotype can modify B-cell responses by differential activation of TH1 subsets. Diminished humoral immunity, as was observed with the E1 and E4 deletion vectors in lung, is indeed desired in applications of gene therapy where readministration of the vector is necessary.     

Induction of central tolerance by intrathymic inoculation of adenoviral antigens into the host thymus permits long-term gene therapy in Gunn rats

Recombinant adenoviruses are highly efficient at transferring foreign genes in vivo. However, duration of gene expression is limited by the host antiviral immune response which precludes expression upon viral readministration. We tested the feasibility of prolonging gene expression by induction of central tolerance to adenoviral antigens in bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient Gunn rats. Tolerance was induced by intraperitoneal injection of antilymphocyte serum, followed by intrathymic inoculation of one of the following: a recombinant adenovirus (Ad), adenovirus human UDP-glucuronosyltransferase (Ad-hBUGT1) carrying the hBUGT1 gene; a protein extract of the same virus; or viral infected hepatocytes. Controls received intrathymic injections of normal saline. After 12 d all groups were injected intravenously with 5 x 10(9) pfu of either Ad-hBUGT1 or adenovirus beta-galactosidase (Ad-LacZ) (expressing the Escherichia coli beta-galactosidase [LacZ] gene). In all three groups of tolerized rats, hBUGT1 was expressed in the liver after administration of Ad-hBUGT1, with glucuronidation of biliary bilirubin of above 95%. Serum bilirubin levels decreased from 7.2 to 1.8 mg/dl within 1 wk and remained low for 7 wk. Similar findings were observed following repeat injections given on days 45 and 112. In control rats serum bilirubin levels were reduced for only 4 wk, and viral readministration was ineffective. In all tolerized groups, but not in controls, there was a marked inhibition of appearance of neutralizing antibodies and cytotoxic lymphocytes against the recombinant adenovirus. Injection of wild type adenovirus-5 (Ad5) into the tolerized rats elicited a wild type-specific cytotoxic lymphocyte response. This is the first demonstration of Ad-directed long-term correction of an inherited metabolic disease following central tolerization with thymic antigen.     

Strain-specific neutralization of human cytomegalovirus isolates by human sera

Induction of an effective antibody response against human cytomegalovirus (HCMV) is an important defense mechanism since it is potentially capable of neutralizing infectious viruses. We have analyzed the extent of HCMV strain-specific neutralization capacity in human sera. Nine recent HCMV isolates and their corresponding sera were investigated in cross-neutralization assays. We observed differences, independent of the overall neutralization capacity, in the 50% neutralization titers of the sera against individual strains, differences that ranged from 8-fold to more than 60-fold. For one isolate, complete resistance to neutralization by two human sera was observed. The neutralization capacity of human sera was not influenced by the presence of various concentrations (up to 100-fold excess) of noninfectious envelope glycoproteins, an inherent contamination of virus preparations from recent HCMV isolates. This indicated that the decisive parameter for neutralization is the titer of the neutralizing antibodies and that neutralization is largely independent of the concentration of virus. Analysis with transplant patients revealed that during primary infection strain-specific and strain-common antibodies are produced asynchronously. Thus, our data demonstrate that the induction of strain-specific neutralizing antibodies is a common event during infection with HCMV and that it might have important implications for the course of the infection and the development of anti-HCMV vaccines.     

Combinatorial blockade of calcineurin and CD28 signaling facilitates primary and secondary therapeutic gene transfer by adenovirus vectors in dystrophic (mdx) mouse muscles

Recombinant adenovirus vectors (AdV) have been considered a potential vehicle for performing gene therapy in patients suffering from Duchenne muscular dystrophy but are limited by a cellular and humoral immune response that prevents long-term transgene expression as well as effective transduction after AdV readministration. Conventional immunosuppressive agents such as cyclosporine and FK506, which act by interfering with CD3-T-cell receptor-mediated signaling via calcineurin, are only partially effective in reversing these phenomena and may also produce substantial organ toxicity. We hypothesized that activation of redundant T-cell activation pathways could limit the effectiveness of these drugs at clinically tolerable doses. Therefore, we have tested the ability of immunomodulatory immunoglobulins (Ig) with different modes of action to facilitate AdV-mediated gene transfer to adult dystrophic (mdx) mice. When used in isolation, immunomodulatory Ig (anti-intercellular adhesion molecule-1, anti-leukocyte function-associated antigen-1, anti-CD2, and CTLA4Ig) were only mildly effective in mitigating cellular and/or humoral immunity against adenovirus capsid proteins and the therapeutic transgene product, dystrophin. However, the combination of FK506 plus CTLA4Ig abrogated the immune response against adenovirus proteins and dystrophin to a degree not achievable with the use of either agent alone. At 30 days after AdV injection, >90% of myofibers could be found to express dystrophin with little or no evidence of a cellular immune response against transduced fibers. In addition, the humoral immune response was markedly suppressed, and this was associated with increased transduction efficiency following vector readministration. These data suggest that by facilitating both primary and secondary transduction after AdV administration, combined targeting of CD3-T-cell receptor-mediated signaling via calcineurin and the B7:CD28 costimulatory pathway could greatly increase the potential utility of AdV-mediated gene transfer as a therapeutic modality for genetic diseases such as Duchenne muscular dystrophy that will require long-term transgene expression and repeated vector delivery.     

Broad cross-neutralizing activity in serum is associated with slow progression and low risk of transmission in primate lentivirus infections

Sera from human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2)-infected humans were tested with autologous (from the same individual) and heterologous (from other individuals) virus isolates in a neutralization assay. Similarly, sera from experimentally simian immunodeficiency virus (SIVsm from sooty mangabey) or HIV-2SBL6669-infected cynomolgus macaques were tested for neutralizing activity against autologous and heterologous reisolates. In the neutralization assay, the virus dose ranged between 10-75 50% infectious dose (ID50), sera were used in five 2- or 4-fold dilutions, beginning with 1:20, and human peripheral blood mononuclear cells (PBMCs) served as target cells. The readout of the 7-day assay was a HIV-1 or HIV-2 antigen enzyme-linked immunosorbent assay (ELISA). Our results show that SIVsm-inoculated monkeys who develop early immunodeficiency lack serum neutralizing activity or develop a neutralizing antibody response with narrow specificity. Long survival is associated with the ability to neutralize several autologous and heterologous SIVsm reisolates. Infection of macaques with HIV-2SBL6669 did not cause disease within the 5 years observation time and elicited a broadly cross-reactive neutralizing antibody response, including neutralization of other, independently obtained, HIV-2 isolates. In HIV-1-infected humans, neutralizing antibodies can only be detected in up to 50% of cases. Neutralizing activity, whenever present, may show a broad specificity, that is, neutralization may occur across genetic subtypes. Presence of broadly cross-reactive neutralizing antibodies is associated with a lower risk of HIV-1 (subtype B) transmission both from mother to child and sexually from male to female. Unlike HIV-1 infection, serum neutralizing activity is regularly present in HIV-2 infection. In view of the differences between HIV-1 and HIV-2 pathogenesis, we suggest that an effective neutralizing antibody response may contribute to a delay in disease progression and to a decrease in risk of transmission.     

Efficient, repeated adenovirus-mediated gene transfer in mice lacking both tumor necrosis factor alpha and lymphotoxin alpha

The efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, and the related cytokine lymphotoxin alpha (LTalpha), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-alpha/LTalpha-/-), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-alpha/LTalpha-/- and TNF-alpha/LTalpha+/- mice versus TNF-alpha/LTalpha+/+ mice links antibody levels to TNF-alpha/LTalpha gene dosage. Due to the absence of neutralizing antibodies, the TNF-alpha/LTalpha knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-alpha in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.     

Neutralization of HIV-1 primary isolates by polyclonal and monoclonal human antibodies

To examine antibody-mediated neutralization of HIV-1 primary isolates in vitro, we tested sera and plasma from infected individuals against four clade B primary isolates. These isolates were analyzed further for neutralization by a panel of several human anti-HIV-1 mAb in order to identify the neutralizing epitopes of these viruses. Each of the HIV-1+ serum and plasma specimens tested had neutralizing activities against one or more of the four primary isolates. Of the three individual sera, one (FDA-2) neutralized all of the four isolates, while the other two sera were effective against only one virus. The pooled plasma and serum samples reacted broadly with these isolates. Based on the neutralizing activities of the mAb panel, each virus isolate exhibited a distinct pattern of reactivity, suggesting antigenic diversity among clade B viruses. Neutralizing epitopes were found in the V3 loop and CD4-binding domain of gp120, as well as near the transmembrane region (cluster II epitope) of gp41. A mAb directed to the cluster I epitope of gp41 near the immunodominant disulfide loop weakly neutralized one primary isolate. None of the mAb in the panel affected one primary isolate, US4, although this virus was sensitive to neutralization by some of the polyclonal antibody specimens. This isolate was also resistant to neutralization by a cocktail of 10 mAb, most of which individually inhibited at least one of the other three viruses tested. These results suggest that neutralizing activity for this latter virus is present in certain HIV-1+ sera/plasma, but is not exhibited by the mAb in the panel. Thus, effective neutralizing antibodies against primary isolates can be generated by humans upon exposure to HIV-1, but not all of these antigenic specificities are represented in a large panel of human anti-HIV-1 mAb.     

Study of the V3 loop as a target epitope for antibodies involved in the neutralization of primary isolates versus T-cell-line-adapted strains of human immunodeficiency virus type 1

Previous studies characterized the third variable (V3) loop of the envelope gp120 as the principal neutralizing determinant for laboratory T-cell-line-adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1). However, primary viruses isolated from infected individuals are more refractory to neutralization than TCLA strains, suggesting that qualitatively different neutralizing antibodies may be involved. In this study, we investigated whether the V3 loop constitutes a linear target epitope for antibodies neutralizing primary isolates. By using peptides representative of the V3 regions of various primary isolates, an early, relatively specific and persistent antibody response was detected in sera from HIV-infected patients. To assess the relationship between these antibodies and neutralization, the same peptides were used in competition and depletion experiments. Addition of homologous V3 peptides led to a competitive inhibition in the neutralization of the TCLA strain HIVMN/MT-4 but had no effect on the neutralization of the autologous primary isolate. Similarly, the removal of antibodies that bind to linear V3 epitopes resulted in a loss of HIVMN/MT-4 neutralization, whereas no decrease in the autologous neutralization was measured. The different roles of V3-specific antibodies according to the virus considered were thereby brought to light. This confirmed the involvement of V3 antibodies in the neutralization of a TCLA strain but emphasized a more pronounced contribution of either conformational epitopes or epitopes outside the V3 loop as targets for antibodies neutralizing primary HIV-1 isolates. This result underlines the need to focus on new vaccinal immunogens with epitopes able to induce broadly reactive and efficient antibodies that neutralize a wide range of primary HIV-1 isolates.     

Immunizing patients with metastatic melanoma using recombinant adenoviruses encoding MART-1 or gp100 melanoma antigens

BACKGROUND: The characterization of the genes encoding melanoma-associated antigens MART-1 or gp100, recognized by T cells, has opened new possibilities for the development of immunization strategies for patients with metastatic melanoma. With the use of recombinant adenoviruses expressing either MART-1 or gp100 to immunize patients with metastatic melanoma, we evaluated the safety, immunologic, and potential therapeutic aspects of these immunizations. METHODS: In phase I studies, 54 patients received escalating doses (between 10(7) and 10(11) plaque-forming units) of recombinant adenovirus encoding either MART-1 or gp100 melanoma antigen administered either alone or followed by the administration of interleukin 2 (IL-2). The immunologic impact of these immunizations on the development of cellular and antibody reactivity was assayed. RESULTS: Recombinant adenoviruses expressing MART-1 or gp100 were safely administered. One of 16 patients with metastatic melanoma receiving the recombinant adenovirus MART-1 alone experienced a complete response. Other patients achieved objective responses, but they had received IL-2 along with an adenovirus, and their responses could be attributed to the cytokine. Immunologic assays showed no consistent immunization to the MART-1 or gp100 transgenes expressed by the recombinant adenoviruses. High levels of neutralizing antibody were found in the pretreatment sera of the patients. CONCLUSIONS: High doses of recombinant adenoviruses could be safely administered to cancer patients. High levels of neutralizing antibody present in patients' sera prior to treatment may have impaired the ability of these viruses to immunize patients against melanoma antigens.     

Extensive cross-reactivity of adenovirus-specific cytotoxic T cells

Although adenovirus is a major source of morbidity for immunocompromised individuals and a popular vector for gene therapy, little is known about the cellular immune responses it evokes in humans. Initial trials using adenovirus vectors have been disappointing, probably owing both to a preexisting immune response to Ad2 and Ad5, the most commonly used vector backbones, and to a response to the transgene. The former problem might be overcome by switching from the common type C adenoviruses, of which Ad2 and Ad5 are members, to other less common serotypes. Evidence for the feasibility of this approach has been provided by a rat model system. However, its success in humans depends on there being no immunological cross-reactivity between groups at the humoral or cellular level. Here, we examine the cross-reactivity of the cellular immune response to adenovirus in a human system, and find that human cytotoxic T lymphocytes (CTLs) prepared in vitro against an adenovirus from two of the six subgroups can lyse cells infected with adenoviruses from the other subgroups. Hence, the proposed use of adenovirus vectors from uncommon subgroups to evade memory immune response to subgroup C adenoviruses may not be successful. However, this same cross-reactivity indicates that adoptive transfer of CTLs generated in vitro against one adenovirus serotype may protect immunocompromised patients from infections by adenoviruses of all serotypes.     

Successful readministration of adeno-associated virus vectors to the mouse lung requires transient immunosuppression during the initial exposure

The airway is an important target for gene transfer to treat cystic fibrosis and other diseases that affect the lung. We previously found that marker gene expression did not persist in the bronchial epithelium following adeno-associated virus (AAV) vector administration to the rabbit lung. In an attempt to promote continued expression, we tested repeat vector administration, but no additional transduction was observed, and the block to transduction correlated with the appearance of neutralizing antibodies to the viral capsid. Here we show that mice exhibit a similar response but that treatment with anti-CD40 ligand antibody (MR1) and a soluble CTLA4-immunoglobulin fusion protein (CTLA4Ig) at the time of primary AAV vector exposure allowed successful repeat transduction and prevented production of neutralizing antibodies. We also tested the possibility that an immune response caused the loss of marker-positive cells in the epithelial population in rabbits by evaluating AAV vector expression in immunocompetent and immunodeficient mice. In contrast to results in rabbits, marker protein expression persisted in the lung in both groups of mice. AAV vector transduction occurred in alveolar cells, airway epithelial cells, and smooth muscle cells, and vector expression persisted for at least 8 months. Although data on persistence of AAV vector expression in the human lung are not available, it is likely that repeat transduction will be necessary either due to loss of expression or to the need for repeat administration to deliver effective amounts of AAV vectors. Results presented here indicate that transient immunosuppression will allow such repeat vector treatment of the lung.     

Characterization of human serotype 1 astrovirus-neutralizing epitopes

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Comparative analysis of humoral immune responses to HIV type 1 envelope glycoproteins in mice immunized with a DNA vaccine, recombinant Semliki Forest virus RNA, or recombinant Semliki Forest virus particles

The Semliki Forest virus (SFV) system seems to be a useful new approach for generating effective immune responses against HIV-1 in animal models. We evaluated this system by comparing the humoral immune responses raised in mice immunized against the HIV-1 envelope with the SFV system, a DNA vaccine, and a recombinant Env glycoprotein. gp160 ELISA antibody titers (204,800) were highest in the sera from mice immunized with recombinant Semliki Forest virus particles. These sera contained antibodies to the CD4-binding site and recognized linear epitopes on gp120 and gp41 that were also recognized by a pool of sera from HIV1-infected individuals. This demonstrates that the HIV-1 envelope produced in vivo by the SFV system does not fold aberrantly. A low level of neutralizing antibodies against the HIV-1LAI strain was also detected in the serum of one mouse immunized with recombinant SFV particles, suggesting that booster injections should be given to achieve a more effective immune response. SFV recombinant particles induced the strongest humoral responses to the HIV-1 envelope of all the potential HIV env vaccines tested.     

Identification of a novel c-DNA overexpressed in Fanconi''s anemia fibroblasts partially homologous to a putative L-3-phosphoserine-phosphatase

Glycoprotein (G) of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) contains several neutralizing epitopes. However, recombinant G protein never matches intact viral particles for immunogenicity. DNA immunization offers the possibility to deliver the antigen through the cellular machinery, thus mimicking natural infection. We constructed pCDNA gVHS and pCDNA gIHN plasmids with the G gene of VHSV and IHNV under the control of the CMV promoter, and we tested the plasmids for the accurate G protein expression prior to their use in fish immunization. Following intramuscular injection to adult rainbow trout, plasmid DNA was found inside the muscle cells shortly after injection and was still present 45 days later. mRNA of the G protein was detected in muscle tissue extracts, and the G protein was found within muscle cells at the site of injection. This resulted in the synthesis of high levels of specific neutralizing and protective antibodies. Fish injected with pCDNA gVHS and pCDNA gIHN in combination responded similarly to fish receiving one recombinant plasmid. In addition to the elicitation of a strong humoral response, DNA immunization was able to activate specialized cells of the immune system as well as nonspecific defense mechanisms, since mRNAs of MHC class II and Mx were strongly activated at the site of injection.     

Poly (lactic-glycolic) acid copolymer encapsulation of recombinant adenovirus reduces immunogenicity in vivo

Adenovirus-mediated gene transfer has application to the treatment of diseases of the central nervous system. We demonstrate that a limitation to its use in vivo is an inability to redose to the brain. We show that one factor inhibiting re-dosing is the development of neutralizing anti-adenoviral antibodies. Encapsulation of recombinant adenovirus vectors in poly(lactic/glycolic acid) (PLGA) copolymer enables infection in vitro, in the presence of neutralizing antibodies and results in the release of viable virus for over 100 h. Importantly, encapsulated adenovirus also shows diminished immunogenicity in vivo. Mice immunized with encapsulated recombinant adenoviral vectors show a greater than 45-fold reduction in anti-adenovirus titers relative to non-encapsulated vectors. An extended release formulation of adenovirus that reduces viral immunogenicity and sequesters the viral particle form antibody exposure may improve in vivo efficacy.     

Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected to elicit antibodies preferentially neutralizing mutant variants of HIV-BRU lacking the N306 glycan. Therefore, two guinea pigs were immunized with monomeric wild-type HIV-BRU gp120 possessing the N306 glycan and immune sera were tested for neutralization against target viruses HIV-BRU, -A308, and -A308T321. HIV-A308 and HIV-A308T321 lack the N306 glycan; HIV-A308T321 contains an additional mutation at the tip of V3 rendering it resistant to MAb binding at this epitope. Both immune sera preferentially neutralized the two mutant virus variants lacking the N306 glycan, with a 10- to 20-fold increase in neutralization titer compared with the wild-type HIV-BRU. Thus, immunization with monomeric HIV-BRU gp120 elicited antibodies preferentially neutralizing HIV variants lacking the N306 glycan. In addition to antibodies directed against the tip of V3, other antibodies directed against epitopes shielded by the N306 glycan on the envelope oligomer were elicited by the immunization, as demonstrated by the ability of the immune sera to neutralize HIV-A308T321. One such epitope was overlapping the NEA-9284 epitope located at the amino-terminal flank of the V3 loop. Our results demonstrate that monomeric gp120 contains immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation.