THE rhGM-CSF–EPO HYBRID PROTEIN MEN 11300 INDUCES ANTI-EPO ANTIBODIES AND SEVERE ANAEMIA IN RHESUS MONKEYS

A recombinant human GM-CSF-EPO hybrid protein named MEN 11300 was administered biweekly for a total of 6 weeks to rhesus monkeys in order to evaluate its pharmacokinetic behaviour, tolerability and immunogenicity. In this primate species a strong antibody response was induced which neutralized the in vitro biological activity of human EPO while no antibody response could be detected against human GM-CSF. A severe drop in reticulocyte counts at approximately 2 weeks after initiation of treatment was followed by a dramatic decrease in the number of erythrocytes. No effects were observed on GM-CSF-dependent hematopoietic lineages and the clinical chemistry analyses did not reveal signs of general toxicity. Reticulocyte and erythrocyte counts started to recover 3–4 weeks after discontinuation of treatment in concert with a decline in anti-EPO antibody titres. Nevertheless, cell numbers remained below basal levels up to 50 days after the last MEN 11300 administration. Haematological impairment indicates that the administration to non-human primate of human EPO fused to human GM-CSF, induces neutralizing autoantibodies to the self EPO. Present data do not allow prediction of the immunogenic potential of the fusion protein in humans and a dose-escalating phase I study should be addressed to investigate the safety of the product.     

The rhGM-CSF-EPO hybrid protein MEN 11300 induces anti-EPO antibodies and severe anaemia in rhesus monkeys

A recombinant human GM-CSF-EPO hybrid protein named MEN 11300 was administered biweekly for a total of 6 weeks to rhesus monkeys in order to evaluate its pharmacokinetic behaviour, tolerability and immunogenicity. In this primate species a strong antibody response was induced which neutralized the in vitro biological activity of human EPO while no antibody response could be detected against human GM-CSF. A severe drop in reticulocyte counts at approximately 2 weeks after initiation of treatment was followed by a dramatic decrease in the number of erythrocytes. No effects were observed on GM-CSF-dependent hematopoietic lineages and the clinical chemistry analyses did not reveal signs of general toxicity. Reticulocyte and erythrocyte counts started to recover 3-4 weeks after discontinuation of treatment in concert with a decline in anti-EPO antibody titres. Nevertheless, cell numbers remained below basal levels up to 50 days after the last MEN 11300 administration. Haematological impairment indicates that the administration to non-human primate of human EPO fused to human GM-CSF, induces neutralizing autoantibodies to the self EPO. Present data do not allow prediction of the immunogenic potential of the fusion protein in humans and a dose-escalating phase I study should be addressed to investigate the safety of the product.     

Structural characterization and independent folding of a chimeric glycoprotein comprising granulocyte-macrophage colony stimulating factor and erythropoietin sequences

MEN 11300 is a hybrid glycoprotein of 297 amino acids obtained by fusion of the cDNA encoding GM-CSF with the cDNA encoding EPO followed by transfection of the hybrid gene into CHO cells. The oligonucleotide construct incorporated a spacing sequence between the two individual cDNAs which encodes eight amino acids constituting a linker peptide intended to separate the GM-CSF and EPO moieties. The recombinant MEN 11300 protein was submitted to a detailed structural characterization including the verification of the entire amino acid sequence, the assignment of the disulfide bridges pattern, the identification of the glycosylation sites and the definition of the glycosidic moiety, including site-specificity. Partial processing of the C-terminal Arg residue and the occurrence of N-glycosylation sites at Asn27, Asn155, Asn169, Asn214 were established. Moreover, O-glycosylation at Ser257 and at the N-terminal region was also detected. A large heterogeneity was observed in the N-glycans due to the presence of differently sialylated and fucosylated branched complex type oligosaccharides whereas O-linked glycans were constituted by GalGalNAc chains with a different number of sialic acids. The disulfide bridges pattern was established by direct FABMS analysis of the proteolytic digests or by ESMS analysis of HPLC purified fractions. Pairing of the eight cysteine residues resulted in Cys54-Cys96, Cys88-Cys121, Cys138-Cys292, and Cys160-Cys164. This S-S bridges pattern is identical to that occurring in the individual natural GM-CSF and EPO, thus showing that the two protein moieties in MEN 11300 can independently acquire their native three-dimensional structure.      

Induction of human immunodeficiency virus neutralizing antibodies using fusion complexes

Human immunodeficiency virus-1 (HIV-1) infects cells by membrane fusion that is mediated by the envelope proteins gp120/gp41 and the cellular receptors CD4 and CCR5. During this process, some conserved viral epitopes are temporarily exposed and may induce a neutralizing antibody response when fixed in the fusogenic conformation. These transient structures are conserved and may be effective antigens for use in an anti-HIV-1 vaccine. In this study we tested different conditions of preparation of fusion complexes inducing neutralizing antibodies against both R5 and X4 tropic HIV-1 strains. Cell lines expressing HIV-1 gp120/gp41 and CD4-CCR5 were prepared and conditions for producing fusion complexes were tested. Complexes produced at different temperature and fixative combinations were used to immunize mice. Results indicated that (a) fusion complexes prepared at either 21 degrees C, 30 degrees C or 37 degrees C were immunogenic and induced neutralizing antibodies against both R5 and X4 HIV-1 heterologous isolates; (b) after extensive purification of antibodies there was no cytotoxic effect; (c) complexes prepared at 37 degrees C were more immunogenic and induced higher titers of neutralizing antibodies than complexes prepared at either 21 degrees C or 30 degrees C; (d) the fixative used did not affect the titer of neutralizing antibodies except for glutaraldehyde which was ineffective; (e) the neutralizing activity was retained after CD4-CCR5 antibody removal. The production of higher titers of neutralizing antibody with fusion complexes prepared at 37 degrees C, as compared to lower temperatures, may be related to the induction of antibodies against many different conformation intermediates that subsequently act synergistically at different steps in the fusion process.     

Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure

Recombinant adeno-associated virus type 2 (AAV) is a common vector used in human gene therapy protocols. We characterized the humoral immune response to AAV and observed that 80% of normal human subjects have anti-AAV antibodies and that 18% have neutralizing antibodies. To analyze the effect of neutralizing antibodies on AAV readministration, we attempted to deliver recombinant AAV expressing human factor IX (AAV-hFIX) intraportally into the livers of mice which had been preexposed to AAV and shown to harbor a neutralizing antibody response. While all naive control mice expressed hFIX following administration of AAV-hFIX, none of the mice with preexisting immunity expressed hFIX, even after transient immunosuppression at the time of the second administration with anti-CD4 or anti-CD40L antibodies. This suggests that preexisting immunity to AAV, as measured by a neutralizing antibody response, may limit AAV-mediated gene delivery. Using human sera in an enzyme-linked immunosorbent assay for AAV and a capsid peptide scan library to block antibody binding, we mapped seven regions of the AAV capsid containing immunogenic epitopes. Using pools of these peptides to inhibit the binding of neutralizing antibodies, we have identified a subset of six peptides which potentially reconstitute a single neutralizing epitope. This information may allow the design of reverse genetic approaches to circumvent the preexisting immunity that can be encountered in some individuals.     

Structure-selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern

Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to affect the quality of the antibody response focusing it to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and three variants of concern (VOC), B.1.351, B.1.617.2, and BA.1. We demonstrate that immunogens built on structure selection can be used to influence the quality of the antibody response by focusing it to a conserved site of vulnerability shared between wildtype virus and VOCs, resulting in neutralizing antibodies across variants.     

Variability of Human Systemic Humoral Immune Responses to Adenovirus Gene Transfer Vectors Administered to Different Organs

Administration of adenovirus (Ad) vectors to immunologically naive experimental animals almost invariably results in the induction of systemic anti-Ad neutralizing antibodies. To determine if the human systemic humoral host responses to Ad vectors follow a similar pattern, we evaluated the systemic (serum) anti-Ad serotype 5 (Ad5) neutralizing antibodies in humans after administration of first generation (E1(-) E3(-)) Ad5-based gene transfer vectors to different hosts. AdGVCFTR.10 (carrying the normal human cystic fibrosis [CF] transmembrane regulator cDNA) was sprayed (8 x 10(7) to 2 x 10(10) particle units [PU]) repetitively (every 3 months or every 2 weeks) to the airway epithelium of 15 individuals with CF. AdGVCD.10 (carrying the Escherichia coli cytosine deaminase gene) was administered (8 x 10(8) to 8 x 10(9) PU; once a week, twice) directly to liver metastasis of five individuals with colon cancer and by the intradermal route (8 x 10(7) to 8 x 10(9) PU, single administration) to six healthy individuals. AdGVVEGF121.10 (carrying the human vascular endothelial growth factor 121 cDNA) was administered (4 x 10(8) to 4 x 10(9.5) PU, single administration) directly to the myocardium of 11 individuals with ischemic heart disease. Ad vector administration to the airways of individuals with CF evoked no or minimal serum neutralizing antibodies, even with repetitive administration. In contrast, intratumor administration of an Ad vector to individuals with metastatic colon cancer resulted in a robust antibody response, with anti-Ad neutralizing antibody titers of 10(2) to >10(4). Healthy individuals responded to single intradermal Ad vector variably, from induction of no neutralizing anti-Ad antibodies to titers of 5 x 10(3). Likewise, individuals with ischemic heart disease had a variable response to single intramyocardial vector administration, ranging from minimal neutralizing antibody levels to titers of 10(4). Evaluation of the data from all trials showed no correlation between the peak serum neutralizing anti-Ad response and the dose of Ad vector administered (P > 0.1, all comparisons). In contrast, there was a striking correlation between the peak anti-Ad5 neutralizing antibody levels evoked by vector administration and the level of preexisting anti-Ad5 antibodies (P = 0.0001). Thus, unlike the case for experimental animals, administration of Ad vectors to humans does not invariably evoke a systemic anti-Ad neutralizing antibody response. In humans, the extent of the response is dictated by preexisting antibody titers and modified by route of administration but is not dose dependent. Since the extent of anti-Ad neutralizing antibodies will likely modify the efficacy of administration of Ad vectors, these observations are of fundamental importance in designing human gene therapy trials and in interpreting the efficacy of Ad vector-mediated gene transfer.     

Immunologic priming with recombinant hepatitis A virus capsid proteins produced in Escherichia coli.

Hepatitis A virus capsid proteins (VP0, VP3, and VP1) have been synthesized in Escherichia coli for use in antigenic and immunogenic analyses. Rabbits immunized with each of these individual recombinant capsid proteins developed a rapid neutralizing antibody response when subsequently challenged with a subimmunogenic dose of whole virus.     

Immunogenic and functional organization of hepatitis C virus (HCV) glycoprotein E2 on infectious HCV virions

Development of full-length hepatitis C virus (HCV) RNAs replicating efficiently and producing infectious cell-cultured virions, HCVcc, in hepatoma cells provides an opportunity to characterize immunogenic domains on viral envelope proteins involved in entry into target cells. A panel of immunoglobulin G1 human monoclonal antibodies (HMAbs) to three immunogenic conformational domains (designated A, B, and C) on HCV E2 glycoprotein showed that epitopes within two domains, B and C, mediated HCVcc neutralization, whereas HMAbs to domain A were all nonneutralizing. For the neutralizing antibodies to domain B (with some to conserved epitopes among different HCV genotypes), the inhibitory antibody concentration reducing HCVcc infection by 90%, IC90, ranged from 0.1 to 4 microg/ml. For some neutralizing HMAbs, HCVcc neutralization displayed a linear correlation with an antibody concentration between the IC50 and the IC90 while others showed a nonlinear correlation. The differences between IC50/IC90 ratios and earlier findings that neutralizing HMAbs block E2 interaction with CD81 suggest that these antibodies block different facets of virus-receptor interaction. Collectively, these findings support an immunogenic model of HCV E2 having three immunogenic domains with distinct structures and functions and provide added support for the idea that CD81 is required for virus entry.     

The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity.

The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes. We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity. For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli. All purified hybrid HBc/pre-S polyproteins were particulate. Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity. In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc. Predictably, HBcAg-specific immunogenicity was also reduced. With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic. A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic. A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic. Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response. Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.     

Treatment-induced antibodies to interleukin-2

Interleukin-2 (IL-2) is a 15 kDa glycoprotein with proven activity as an immune stimulant in the treatment of malignant disorders, congenital and acquired immune deficiencies, infectious disorders, and as an adjuvant to vaccines. Both natural and recombinant type IL-2 preparations have been applied in clinical treatment trials and have turned out to be immunogenic, although to a varying extent. Enzyme immunoassays and western blotting are standard procedures for the detection of IL-2-binding antibodies, whereas the neutralizing capacity of these antibodies is frequently demonstrated by inhibition of IL-2-dependent cell growth in vitro. The rate of treatment-induced IL-2 antibodies has varied from 0% to 100% in reported trials and frequently exceeded 50% in patients exposed to recombinant IL-2, whereas natural type IL-2 appeared to be little immunogenic. Duration of treatment, cumulative IL-2 dose, and route of IL-2 administration are likely to determine both the rate of seroconversion as well as composition and properties of the anti-IL-2 antibodies. Interleukin-2 antibodies are polyclonal in nature and predominantly composed of IgM and IgG types. Frequently they react with both recombinant and natural IL-2 types. As a rule, neutralizing IL-2 antibodies are detected in serum samples with high IL-2-binding titers and are recognized later than their non-neutralizing predecessors. Neutralization in vitro, however, does not predict neutralization in vivo, and there are very rare patients with documented, antibody-mediated loss of response to IL-2 treatment. More frequently, IL-2 antibodies will limit the expression of IL-2-dependent proteins in vivo, but the opposite has also been observed. Although the precise mechanism of antibody induction by IL-2 is unknown, immunogenicity of some drug formulations rather than polyclonal B-cell activation appears to play a critical role. Approaches aiming at limiting the immunogenicity of IL-2 preparations are discussed, and strategies how to recognize and circumvent antibody-mediated IL-2 resistance are presented.     

Dissection of Antibody Specificities Induced by Yellow Fever Vaccination

The live attenuated yellow fever (YF) vaccine has an excellent record of efficacy and one dose provides long-lasting immunity, which in many cases may last a lifetime. Vaccination stimulates strong innate and adaptive immune responses, and neutralizing antibodies are considered to be the major effectors that correlate with protection from disease. Similar to other flaviviruses, such antibodies are primarily induced by the viral envelope protein E, which consists of three distinct domains (DI, II, and III) and is presented at the surface of mature flavivirions in an icosahedral arrangement. In general, the dominance and individual variation of antibodies to different domains of viral surface proteins and their impact on neutralizing activity are aspects of humoral immunity that are not well understood. To gain insight into these phenomena, we established a platform of immunoassays using recombinant proteins and protein domains that allowed us to dissect and quantify fine specificities of the polyclonal antibody response after YF vaccination in a panel of 51 vaccinees as well as determine their contribution to virus neutralization by serum depletion analyses. Our data revealed a high degree of individual variation in antibody specificities present in post-vaccination sera and differences in the contribution of different antibody subsets to virus neutralization. Irrespective of individual variation, a substantial proportion of neutralizing activity appeared to be due to antibodies directed to complex quaternary epitopes displayed on the virion surface only but not on monomeric E. On the other hand, DIII-specific antibodies (presumed to have the highest neutralizing activity) as well as broadly flavivirus cross-reactive antibodies were absent or present at very low titers. These data provide new information on the fine specificity as well as variability of antibody responses after YF vaccination that are consistent with a strong influence of individual-specific factors on immunodominance in humoral immune responses.     

epitopes immediately below the base of the V3 loop of gp120 as targets for the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals

Epitopes that drive the initial autologous neutralizing antibody response in HIV-1-infected individuals could provide insights for vaccine design. Although highly strain specific, these epitopes are immunogenic, vulnerable to antibody attack on infectious virus, and could be involved in the ontogeny of broadly neutralizing antibody responses. To delineate such epitopes, we used site-directed mutagenesis, autologous plasma samples, and autologous monoclonal antibodies to map the amino acid changes that led to escape from the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals. Additional mapping of the epitopes was accomplished by using alanine scanning mutagenesis. Escape in the two individuals occurred by different pathways, but the responses in both cases appeared to be directed against the same region of gp120. In total, three amino acid positions were identified that were independently associated with autologous neutralization. Positions 295 and 332 are located immediately before and after the N- and C-terminal cysteines of the V3 loop, respectively, the latter of which affected an N-linked glycan that was critical to the neutralization epitope. Position 415 affected an N-linked glycan at position 413 in the C terminus of V4 that might mask epitopes near the base of V3. All three sites lie in close proximity on a four-stranded antiparallel sheet on the outer domain of gp120. We conclude that a region just below the base of the V3 loop, near the coreceptor binding domain of gp120, can be a target for autologous neutralization.     

Heterologous epitope-scaffold prime:boosting immuno-focuses B cell responses to the HIV-1 gp41 2F5 neutralization determinant

The HIV-1 envelope glycoproteins (Env) gp120 and gp41 mediate entry and are the targets for neutralizing antibodies. Within gp41, a continuous epitope defined by the broadly neutralizing antibody 2F5, is one of the few conserved sites accessible to antibodies on the functional HIV Env spike. Recently, as an initial attempt at structure-guided design, we transplanted the 2F5 epitope onto several non-HIV acceptor scaffold proteins that we termed epitope scaffolds (ES). As immunogens, these ES proteins elicited antibodies with exquisite binding specificity matching that of the 2F5 antibody. These novel 2F5 epitope scaffolds presented us with the opportunity to test heterologous prime:boost immunization strategies to selectively boost antibody responses against the engrafted gp41 2F5 epitope. Such strategies might be employed to target conserved but poorly immunogenic sites on the HIV-1 Env, and, more generally, other structurally defined pathogen targets. Here, we assessed ES prime:boosting by measuring epitope specific serum antibody titers by ELISA and B cell responses by ELISpot analysis using both free 2F5 peptide and an unrelated ES protein as probes. We found that the heterologous ES prime:boosting immunization regimen elicits cross-reactive humoral responses to the structurally constrained 2F5 epitope target, and that incorporating a promiscuous T cell helper epitope in the immunogens resulted in higher antibody titers against the 2F5 graft, but did not result in virus neutralization. Interestingly, two epitope scaffolds (ES1 and ES2), which did not elicit a detectable 2F5 epitope-specific response on their own, boosted such responses when primed with the ES5. Together, these results indicate that heterologous ES prime:boost immunization regimens effectively focus the humoral immune response on the structurally defined and immunogen-conserved HIV-1 2F5 epitope.     

The envelope proteins from purified respiratory syncytial virus protect mice from intranasal virus challenge

A lyophilized subunit vaccine prepared from purified respiratory syncytial virus, which contained the envelope glycoproteins F and G and the nonglycosylated matrix protein VPM, was tested in SJL mice for its ability to protect the lungs of mice from intranasal viral challenge. Initially, the mice were injected subcutaneously with one, two, or three doses of 5 or 25 micrograms of vaccine in 50% complete Freund's adjuvant or with complete Freund's adjuvant or phosphate-buffered saline only. Although none of the mice produced neutralizing serum antibody, three doses of 25 micrograms elicited antibodies to F, G, and VPM. Despite the absence of detectable neutralizing antibodies, the lungs of 93% of the vaccinated mice were protected from intranasal viral challenge. Because the initial protocol did not elicit neutralizing antibodies and a few single-dose animals were not protected, a second vaccine trial was carried out. For these studies the priming dose was increased to 50 micrograms, which was followed, in half the vaccine recipients, by a second dose of 25 micrograms. Mice given the priming dose of vaccine produced antibody to G and showed no neutralizing activity, whereas the mice given two doses of vaccine produced antibodies to G, F, and VPM and also displayed neutralizing activity for respiratory syncytial virus. The lungs of 100% of the vaccine recipients in this trial were protected from intranasal challenge. Although the vaccine elicited antibody to VPM, this response did not correlate with protection. In addition, examination of the sera from unimmunized mice recovering from respiratory syncytial virus infection revealed a serum antibody profile similar to that noted for humans, lacking antibody to VPM. Thus, the data show that a combined glycoprotein subunit vaccine affords complete protection to viral challenge and offers an approach to develop a multivalent subunit vaccine.     

Effect of humoral immune responses on controlling viremia during primary infection of rhesus monkeys with simian immunodeficiency virus

Cellular immune responses mediated by CD8+ lymphocytes exert efficient control of virus replication during primary simian immunodeficiency virus (SIV) infection. However, the role that antibodies may play in the early control of virus replication remains unclear. To evaluate how antibody responses may affect virus replication during primary SIVmac infection, we depleted rhesus monkeys of B cells with anti-CD20 antibody. In normal rhesus monkeys immunized with tetanus toxoid, anti-CD20 treatment and resulting depletion of B cells inhibited the generation of antitetanus antibodies, while tetanus-specific T-cell responses were preserved. During the first 4 weeks after inoculation with SIVmac251, development of SIV-specific neutralizing antibody was delayed, and titers were significantly lower in B-cell-depleted monkeys than control-antibody-treated monkeys. Despite the lower neutralizing antibody titers, the levels of plasma SIV RNA and the linear slope of the decline seen in B-cell-depleted monkeys did not differ from that observed in monkeys treated with control antibody. However, beginning at day 28 after SIV infection, the B-cell-depleted monkeys showed a significant inverse correlation between neutralizing antibody titers and plasma virus level. These results suggest that the rapid decline of peak viremia that typically occurs during the first 3 weeks of infection was not significantly affected by SIV-specific antibodies. However, the inverse correlation between neutralizing antibodies and plasma virus level during the postacute phases of infection suggests that humoral immune responses may contribute to the control of SIV replication.     

FcRn overexpression in mice results in potent humoral response against weakly immunogenic antigen

The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, is active in phagocytosis and delivers antigen for presentation. We have previously shown that transgenic (tg) mice that have been created to overexpress bovine FcRn (bFcRn) demonstrate increased half-life of mouse IgG, significantly increased antigen-specific IgG in serum and augmented expansion of antigen-specific B cells and plasma cells after immunization. One of the interesting questions surrounding this enhanced immune response is whether these tg mice could effectively induce immune response to weakly immunogenic antigens. To address this question, we immunized these bFcRn tg mice with a conserved hemagglutinin subunit 2 (HA2)-based synthetic peptide that was recently found to be effectively targeted by neutralizing antibodies. Using an ELISA system, we found that, whereas wild-type mice showed a weak immune response and developed only a de minimis amount of antibody against the epitope, FcRn overexpressing animals mounted a robust reaction expressed in specific antibody titers on day 28 that continued to rise through day 50. Consistent with our previous data, the enhanced immune response resulting from the FcRn overexpression was also associated with a substantial increase in the number of spleen derived B cells, dendritic cells, granulocytes and plasma cells. Based on this evidence, we propose that tg mice that overexpress bFcRn offer major advantages in monoclonal antibody production because the tg mice would allow the generation of antibodies (hybridomas) to weakly immunogenic antigens that otherwise would be difficult or even impossible to make.Key words: neonatal Fc receptor (FcRn), transgenic mouse, immunogenicity, monoclonal antibody, influenza     

Potential therapeutic use of antibodies directed towards HuIFN-γ

IFN-γ is an important regulator of immune responses and inflammation. Studies in animal models of inflammation, autoimmunity, cancer, transplant rejection and delayed-type hypersensitivity have indicated that administration of antibodies against IFN-γ can prevent the occurrence of diseases or alleviate disease manifestations. Therefore, it is speculated that such antibodies may have therapeutical efficacy in human diseases. Since animal-derived antibodies are immunogenic in patients several strategies are being developed in order to reduce or abolish this human anti-mouse antibody (HAMA) response. In our laboratory, we have constructed a single-chain variable fragment (scFv) derived from a mouse antibody with neutralizing potential for human IFN-γ. A scFv consists of only variable domains tethered together by a flexible linker. The scFv was demonstrated to neutralize the antiviral activity of HuIFN-γin vitro and therefore might be considered as a candidate for human therapy.     

Broadly Neutralizing Immune Responses against Hepatitis C Virus Induced by Vectored Measles Viruses and a Recombinant Envelope Protein Booster

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.     

Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors.