Weak Neutralizing Activity Research Articles

Neutralization sensitivity of HIV-1 subtype B’ clinical isolates from former plasma donors in China

BackgroundHIV-1 subtype B’ isolates have been predominantly circulating in China. Their intra- and inter-subtype neutralization sensitivity to autologous and heterologous plasmas has not been well studied.ResultsTwelve HIV-1 B’ clinical isolates obtained from patients were tested for their intra- and inter-subtype neutralization sensitivity to the neutralization antibodies in the plasmas from patients infected by HIV-1 B’ and CRF07_BC subtypes, respectively. We found that the plasmas from the HIV-1 B’-infected patients could potently neutralize heterologous viruses of subtype B’ with mean ID50 titer (1/x) of about 67, but they were not effective in neutralizing autologous viruses of subtype B’ with mean ID50 titer (1/x) of about 8. The plasmas from HIV-1 CRF07_BC-infected patients exhibited weak inter-subtype neutralization activity against subtype B’ viruses with ID50 titer (1/x) is about 22. The neutralization sensitivity of HIV-1 B’ isolates was inversely correlated with the neutralizing activity of plasmas from HIV-1 B’-infected patients (Spearman’s r = −0.657, P = 0.020), and with the number of potential N-glycosylation site (PNGS) in V1-V5 region (Spearman’s r = −0.493, P = 0.034), but positively correlated with the viral load (Spearman’s r = 0.629, P = 0.028). It had no correlation with the length of V1-V5 regions or the CD4+ T cell count. Virus AH259V has low intra-subtype neutralization sensitivity, it can be neutralized by 17b (IC50: 10μg/ml) and 447-52D (IC50: 1.6μg/ml), and the neutralizing antibodies (nAbs) in plasma AH259P are effective in neutralizing infection by the primary HIV-1 isolates with different subtypes with ID50 titers (1/x) in the range of 32–396.ConclusionsThese findings suggest that the HIV-1 subtype B’ viruses may mutate under the immune pressure, thus becoming resistant to the autologous nAbs, possibly by changing the number of PNGS in the V1-V5 region of the viral gp120. Some of primary HIV-1 isolates are able to induce both intra- and inter-subtype cross-neutralizing antibody responses.

Anti-hepatitis C virus E2 (HCV/E2) glycoprotein monoclonal antibodies and neutralization interference

The suggested HCV escape mechanism consisting in the elicitation of antibody (Ab) subpopulations interfering with the neutralizing activity of other Abs has recently been questioned. In particular, it was originally reported that Abs directed against the 436–447 region (epitope II) of HCV/E2 glycoprotein may interfere with the neutralizing Abs directed against the 412–423 region (epitope I) involved in the binding to CD81. In this paper, we investigate on the molecular features of this phenomenon describing an anti-HCV/E2 monoclonal Ab (mAb) (e509) endowed with a weak neutralizing activity, and whose epitope is centered on epitope II. Interestingly, e509 influenced the potent neutralizing activity of AP33, one of the best characterized anti-HCV/E2 mAb, whereas it did not show any interfering activity against two other broadly neutralizing mAbs (e20 and e137), whose epitopes partially overlap with that of e509 and which possibly displace it from the antigen.These data may give a possible clue to interpret the conflicting studies published to date on the mechanism of interference, suggesting the existence of at least two groups of broadly neutralizing anti-HCV/E2 Abs: (i) those whose epitope is focused on the 412–423 CD81-binding region and whose activity may be hampered by other Abs directed against the 436–447 region, and (ii) those directed against CD81-binding regions but whose epitope contains also residues within the 436–447 region recognized by interfering mAbs, thus competing with them for binding. The conflicting results of previous studies may therefore depend on the relative amount of each of these two populations in the polyclonal preparations used. Overall, a better comprehension of this phenomenon may be of importance in the set up of novel mAb-based anti-HCV therapeutic strategies.

Magnitude and Breadth of a Nonprotective Neutralizing Antibody Response in an Efficacy Trial of a Candidate HIV‐1 gp120 Vaccine

A candidate vaccine consisting of human immunodeficiency virus type 1 (HIV-1) subunit gp120 protein was found previously to be nonprotective in an efficacy trial (Vax004) despite strong antibody responses against the vaccine antigens. Here we assessed the magnitude and breadth of neutralizing antibody responses in Vax004. Neutralizing antibodies were measured against highly sensitive (tier 1) and moderately sensitive (tier 2) strains of HIV-1 subtype B in 2 independent assays. Vaccine recipients were stratified by sex, race, and high versus low behavioral risk of HIV-1 acquisition. Most vaccine recipients mounted potent neutralizing antibody responses against HIV-1(MN) and other tier 1 viruses. Occasional weak neutralizing activity was detected against tier 2 viruses. The response against tier 1 and tier 2 viruses was significantly stronger in women than in men. Race and behavioral risk of HIV-1 acquisition had no significant effect on the response. Prior vaccination had little effect on the neutralizing antibody response that arose after infection. Weak overall neutralizing antibody responses against tier 2 viruses is consistent with a lack of protection in this trial. The magnitude and breadth of neutralization reported here should be useful for identifying improved vaccines.

Characterization and large production of human monoclonal antibodies against the HIV-1 envelope

Peripheral blood lymphocytes from a volunteer immunized with a recombinant vaccinia virus VSC-25 expressing the gp160 env protein of HTLV-IIIB strain and from an asymptomatic HIV-infected individual were immortalized by Epstein-Barr (EBV). Clones which secrete human monoclonal antibodies from the two individuals (DZ, IgG1, lambda and C31, IgG1, kappa) were obtained and were stable for more than 2 years. The two monoclonals were directed against the gp160 env protein of HIV, DZ directed against the gp41 and C31 directed against the gp120. C31 was group-specific, whereas DZ was directed against the HTLV-IIIB and HTLV-RF strains. The epitope recognized by DZ was mapped to the carboxy terminus of the gp41, by expression of HIV DNA fragments in a yeast system and peptide analysis. The C31 epitope was not expressed by the yeast library and not present among the peptides which were tested. Monoclonal antibodies had no inhibitory effect in an HIV-induced cell fusion assay, but DZ showed a weak neutralizing activity against the HTLV-IIIB strain. Cloned EBV-transformed cell lines were fused to a murine myeloma, which allowed the heteromyeloma to be cultivated in serum-free medium. The monoclonal antibodies were produced in large quantity in a hollow-fibre reactor at defined culture conditions and purification procedures.

Monoclonal Antibodies That Bind to Common Epitopes on the Dengue Virus Type 2 Nonstructural-1 and Envelope Glycoproteins Display Weak Neutralizing Activity and Differentiated Responses to Virulent Strains: Implications for Pathogenesis and Vaccines

The abilities of monoclonal antibodies (MAbs) that bind to defined sequential epitopes on the dengue virus (DENV) nonstructural-1 (NS1) glycoproteins to cross-react with epitopes on the DENV envelope (E) glycoproteins were investigated. In this study, some of these MAbs cross-reacted with the DENV type 2 (DENV-2) E glycoprotein and with synthetic peptides representing X-ray crystallographically confirmed surface-exposed regions on this glycoprotein. MAb 1G5.3 cross-reacted with the flavivirus-conserved 101-WGNGCGLFG-109 fusion sequence, the 273-SSGNL-277 DENV-2 hinge region sequence, and the 156-GKHGKEIKIT-165 sequence of virulent DENV-2 strains. MAb 1G5.4-A1-C3 cross-reacted with the 67-NTTTESRCPT-76 and 156-GKHGKEIKIT-165 sequences of virulent DENV-2 strains, the 338-EIMDLDNRHV-347 sequence from a highly virulent DENV-2 (M2) strain, and two epitopes on a virulent DENV-3 strain (288-KMDKLELKG-296 and 323-RVEYRGEDAP-332), which all contained target ELK/KLE-type motifs (underlined). These MAbs showed reduced cross-reactions with the corresponding sequences from weakly pathogenic strains of all four DENV serotypes and had either no (MAb 1G5.4-A1-C3) or weak (MAb 1G5.3) neutralizing activity against them. MAb 1G5.3 more strongly neutralized DENV-2 strains with higher pathogenic capacities, while MAb 1G5.4-A1-C3 showed increasing neutralizing titers against the virulent DENV-3 strain and the moderately virulent and highly virulent (M2) DENV-2 strains. These cross-reactions with the E glycoprotein accord with the observation that MAb 1G5.3 caused dramatic and lethal antibody-enhanced replication (AER) of a DENV-2 strain in vivo. Together with in vivo AER studies of these DENV strains using MAb 1G5.4-A1-C3, these results may account for the increased pathogenic capacities of such strains, which is likely to have important implications for pathogenesis and vaccines.

Production and Characterization of Neutralizing Monoclonal Antibodies Against Human Metapneumovirus F Protein

Human metapneumovirus (hMPV) F protein promotes fusion of viral and cell membranes, and is thought to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. In this paper, the development of two mouse monoclonal antibodies (MAbs) by immunization with hMPVinfected cells is described. Immunofluorescence assay (IFA) using hMPV F protein-expressing cells indicated that two MAbs, designated 1G3 and 9B10, recognized hMPV F protein. Both MAbs were found to be reactive to two groups of hMPV by an IFA using two groups of hMPV-infected cells. The 9B10 mAb had strong neutralizing activity against both groups of hMPV, while the 1G3 MAb had only weak neutralization activity. These results indicate that the hMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Production of MAbs to the hMPV F protein is critical for development of diagnostic techniques, vaccine research, and studies on viral pathogenesis.

Molecular characterization of a major outer capsid protein encoded by the Threadfin aquareovirus (TFV) gene segment 10 (S10)

Genome segment 10 (S10) of Threadfin aquareovirus (TFV) was cloned, sequenced, analyzed and found to be 987 bp long encoding a protein of 298 aa with a predicted molecular mass of 32.0 kDa. The TFV S10 gene possesses terminal motifs, (5' GTTTTA and ATTCATC 3') which are also conserved in the S6 and S11 TFV gene segments. Sequence comparison revealed that the TFV S10 gene was similar to the Striped bass reovirus (SBR) VP7 outer capsid protein (OCP). A conserved putative zinc-finger motif, CCHC, present in the mammalian reovirus (MRV) delta3 protein, was identified in TFV and other aquareovirus VP7 protein. Phylogenetic analysis of the TFV VP7 protein indicated that TFV is closely related to SBR and Chum salmon reovirus (CSV) and possibly belong to the same species Aquareovirus A as SBR and CSV. The TFV VP7 protein was expressed in E. coli, purified and injected into mice. Serum specific antibodies were generated, however, the serum showed weak neutralizing activity. In contrast, co-incubation of this serum with another serum obtained from mice immunized with another OCP encoded by the TFV S6 gene segment resulted in a highly elevated antibody neutralization titer.

Prevalence of neutralizing antibodies to Equine rhinitis A and B virus in horses and man

Equine rhinitis viruses (ERVs) are the causative agents of mild to severe upper respiratory infections in horses worldwide. Immunologically, four serotypes of ERVs have been identified. Equine rhinitis A virus (ERAV) and Equine rhinitis B virus 1 (ERBV1) are the most frequent serotypes in Europe. Both viruses have a broad host range in cultured cells with ERAV being able to infect humans. Since there is neither information on the seroprevalence of ERAV and ERBV1 in Austria nor on the zoonotic potential of ERBV1, we investigated 200 horse and 137 veterinary sera for the presence of neutralizing antibodies relating to ERAV and ERBV1. One hundred and eighty (90%) and 173 (86%) horse sera neutralized ERAV and ERBV1, respectively. In contrast, only four (2.7%) and five (3.6%) human sera showed weak neutralizing activity to ERAV and ERBV1, respectively. These results indicate that ERAV and ERBV1 are widespread in the Austrian horse population; however, the risk of acquiring zoonotic infection among veterinarians appears low.

Comparison of immune responses against foot-and-mouth disease virus induced by fusion proteins using the swine IgG heavy chain constant region or β-galactosidase as a carrier of immunogenic epitopes

Previously, we demonstrated that a fusion protein (Gal-FMDV) consisting of β-galactosidase and an immunogenic peptide, amino acids (141–160)–(21–40)–(141–160), of foot-and-mouth disease virus (FMDV) VP1 protein induced protective immune responses in guinea pigs and swine. We now designed a new potential recombinant protein vaccine against FMDV in swine. The immunogenic peptide, amino acids (141–160)–(21–40)–(141–160) from the VP1 protein of serotype O FMDV, was fused to the carboxy terminus of a swine immunoglobulin G single heavy chain constant region and expressed in Escherichia coli. The expressed fusion protein (IgG-FMDV) was purified and emulsified with oil adjuvant. Vaccination twice at an interval of 3 weeks with the emulsified IgG-FMDV fusion protein induced an FMDV-specific spleen proliferative T-cell response in guinea pigs and elicited high levels of neutralizing antibody in guinea pigs and swine. All of the immunized animals were efficiently protected against FMDV challenge. There was no significant difference between IgG-FMDV and Gal-FMDV in eliciting immunity after vaccination twice in swine. However, when evaluating the efficacy of a single inoculation of the fusion proteins, we found that IgG-FMDV could elicit a protective immune response in swine, while Gal-FMDV only elicited a weak neutralizing activity and could not protect the swine against FMDV challenge. Our results suggest that the IgG-FMDV fusion protein is a promising vaccine candidate for FMD in swine.

Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120–CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the β19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies.

Serologic survey of domestic animals for zoonotic arbovirus infections in the Lacandón Forest region of Chiapas, Mexico.

A serologic survey in domestic animals (birds and mammals) was conducted in four communities located in the Lacandón Forest region of northeastern Chiapas, Mexico, during June 29 to July 1, 2001, with the objective to identify zoonotic arboviruses circulating in this area. We collected 202 serum samples from healthy domestic chickens, geese, ducks, turkeys, horses and cattle. The samples were tested by plaque-reduction neutralization test for antibodies to selected mosquito-borne flaviviruses (family Flaviviridae), including St. Louis encephalitis (SLE), Rocio (ROC), Ilheus (ILH), Bussuquara (BSQ), and West Nile (WN) viruses, and selected alphaviruses (family Togaviridae), including Western equine encephalitis (WEE), Eastern equine encephalomyelitis (EEE), and Venezuelan equine encephalitis (VEE) viruses. Neutralizing antibodies to SLE virus were detected in two (8%) of 26 turkeys, 15 (23%) of 66 cattle, and three (60%) of five horses. Antibodies to VEE virus were detected in 29 (45%) of 65 cattle. Because some of these animals were as young as 2 months old, we demonstrated recent activity of these two viruses. Sub-typing of the VEE antibody responses indicated that the etiologic agents of these infections belonged to the IE variety of VEE, which has been reported from other regions of Chiapas. WN virus-neutralizing antibodies were detected in a single cattle specimen (PRNT(90) = 1:80) that also circulated SLE virus-neutralizing antibodies (PRNT(90) = 1:20), suggesting that WN virus may have been introduced into the region. We also detected weak neutralizing activity to BSQ virus in four cattle and a chicken specimen, suggesting the presence of this or a closely related virus in Mexico. There was no evidence for transmission of the other viruses (ROC, ILH, EEE, WEE) in the study area.

HIV type 1-specific IgG2 antibodies: markers of helper T cell type 1 response and prognostic marker of long-term nonprogression.

The helper T type 1 (Th1) function of CD4(+) T lymphocytes is presumed to be of key importance in host defense against HIV-1. As the production of different antibody isotypes is dependent on this helper T function, we investigated whether HIV-1-specific responses of a particular IgG isotype could be a reliable marker of long-term HIV-1 control. Assessment of the IgG subclass distribution in the plasma of HIV-1-infected patients enrolled in the French prospective Asymptomatic Long-Term (ALT) cohort showed that IgG2 directed against HIV-1 Env gp41 and Gag proteins was associated with low viral load, high CD4(+) lymphocyte count, and weak neutralizing activity. By contrast, levels of anti-Env and anti-Pol IgG1 as well as the magnitude of neutralizing activity were correlated with the viral load and thus merely reflect the level of HIV replication. Furthermore, IgG2 directed against Gag proteins was significantly associated with HIV-1 p24-specific Th1 cell production of interferon gamma and interleukin 2. In multivariate analysis, only two variables, anti-gp41 IgG2 and plasma HIV-1 RNA, were found to be independent prognostic factors of remaining long-term nonprogressive over time. By providing new insight into the nature of an HIV-specific antibody response associated with the control of virus replication, these findings have implications for the design of HIV vaccines.

IMMUNOGENICITY AND SPECIFICITY OF THE CANDIDATE MULTI-EPITOPE-VACCINES AGAINST HIV-1

The failure of some candidate HIV-1 vaccines may result from inducing very weak neutralization activity against representative primary viral isolates. Based on our hypothesis that epitope-vaccine may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, we designed two candidate multi-epitope-vaccines, EP1 [C-G-(ELDKWA-GPGRAFY)2-K] and EP2 (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD), containing three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope protein, and expected them to induce epitope-specific antibodies of predefined epitope-specificity. The two peptides were conjugated to carrier protein bovine serum albumin (BSA) and used for immunization of rabbits. Proteins were purified from the rabbit sera induced by both candidate multi-epitope-vaccines (EP1-BSA and EP2-BSA) through affinity chromatography with epitope-peptide-conjugated sepharose-column, and identified as antibodies in silver-staining and immunoblotting. These antibodies were demonstrated to recognize three neutralizing epitopes on peptides and the recombinant gp41 in ELISA-assay and immunoblotting. These results indicated that both candidate multi-epitope-vaccines could induce high levels of antibodies of predefined epitope-specificity which recognized a few of neutralizing epitopes on peptides and protein, providing experimental evidence for the new strategy to develop an effective neutralizing-antibody-based multi-epitope-vaccine against HIV-1.

Generation of neutralizing human monoclonal antibodies against parvovirus B19 proteins.

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 microgram/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 micrograms/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.

Cloning and Expression of a Single-Chain Antibody Fragment Specific for Foot-and-Mouth Disease Virus

The gene for a single-chain antibody (VHK) to a conformational epitope on the type A12foot-and-mouth disease virus (FMDV) particle was assembled and expressed inEscherichia coli.The VHK, purified from periplasmic extracts immunoprecipitated virus as efficiently as its parental monoclonal antibody (MAb) and exhibited the same binding specificity when tested against a panel of natural and genetically engineered virus particles. The VHK neutralized type A12virus in the presence of goat anti-mouse IgG; however, in the absence of the second antibody, only weak neutralizing activity was detected. Preliminary analysis of the mechanism of viral neutralization indicated that both the MAb and the VHK neutralize by the same mechanism. Small amounts of the VHK allowed infection of cells via Fc receptor-mediated adsorption in the presence of the second antibody. These data represent the first report of a single-chain neutralizing antibody for a picornavirus and provide insights into the mechanisms of viral neutralization and virus uptake.

Construction, Expression, and Immunogenicity of Chimeric HIV-1 Virus-like Particles

The group-specific antigens Pr55gagof human immunodeficiency virus type-1 (HIV-1) self-assemble into noninfectious virus-like particles (VLP) that are released from various eucaryotic cells by budding. Deletion analysis of Pr55gagmutants revealed three domains into which sequences of the third variable domain V3 or the CD4-binding domain of the gp120 external glycoprotein can be inserted without destroying the capacity of the chimeric proteins to assemble to VLP. Immunization of rabbits with different types of purified chimeric VLP without adjuvants raised a strong antibody response to the Pr55gagcarrier component. The magnitude of the antibody response to the inserted gp120 epitopes strictly depended on their position within thegagpolyprotein. These antisera exhibited only weak neutralizing activity. However, BALB/c mice immunized by different routes with different types of chimeric Pr55gag/V3 VLP without adjuvants developed a strong MHC class I (Dd)-restricted, cytolytic CD8+T-cell (CTL) reactivity against a known epitope within the V3 domain. When the recombinant antigen was emulsified in mineral oil (incomplete Freund's adjuvant) or adsorbed in aluminium hydroxide, its immunogenicity for CTL was drastically reduced or completely abrogated. The magnitude of the V3-specific CTL response was not influenced by the position of the V3 domain within the Pr55gag-carrier moiety; the flanking residues, hence, did not influence processing of the exogenous antigen for MHC class I-restricted peptide presentation. These results indicate ways for the rational design and optimal delivery of CTL-stimulating HIV candidate vaccines.

Characterization of monoclonal antibodies recognizing neurotropic Friend murine leukemia virus

We isolated a replication-competent, neurotropic retrovirus (FrC6 virus) and its molecular clone A8 from the NB-tropic Friend murine leukemia virus (FLV) complex. For detection and characterization of the FrC6 and A8 viruses, monoclonal antibodies (MAbs) against the FLV complex were established. Thirty MAbs, each of which reacted with the FLV-producing cell line, were tested for potential neutralizing activities; only two MAbs inhibited the proliferation of the A8 virus. These two MAbs were ineffective or had very weak neutralizing activities toward the non-neurotropic FLV strain clone 57 virus. Further characterization of MAbs by immunoprecipitation revealed that 4 MAbs recognized the envelope protein of the A8 virus. Two of these 4 MAbs recognized the surface glycoprotein gp70, requiring the conformational epitope of the virus for this recognition, while the other two MAbs, which were reactive with the transmembrane protein p15E, were conformation-independent Both of the MAbs against gp70 distinguished neuropathogenic and non-neuropathogenic viruses to some extent, through neutralizing activity or binding activity detected by immunoprecipitation, whereas the two MAbs against p15E reacted with the viruses in a similar manner. Furthermore, one of the MAbs distinguished the viral antigen in the wall of the vacuolation that composes the spongiotic lesion induced by FrC6 viral infection of the brain.

Expression and immunogenicity of an 18-residue epitope of HIV1 gp41 inserted in the flagellar protein of a Salmonella live vaccine

A synthetic oligonucleotide specifying residues 735–752 of the product of the env gene of HIV1 IIIB was inserted by blunt-end ligation at restriction sites in the hypervariabls, antigenically determinant region IV of two flagellin genes. Its integration, in name and correct orientation, into gene fliC(d) in plasmid pLS408 allowed production of functional flagella when the plasmid was placed in a flagellin-negative aroA live-vaccine Salmonella dublin strain, SL5928. Bacteria thus made motile were immobilized and agglutinated by anti-(735–752 peptide) serum; expression was also shown by immunoelectron-microscopy and by Western blot of whole-cell lysates. Enzyme immunoassay (EIA) of sera of mice given three doses by intraperitoneal injection of the live-vaccine strain producing chimeric flagellin, or of concentrated flagella from it showed production of antibody with affinity for the peptide, and in some sera, also for r-gp160. Pooled serum from mice given five i.p. doses of the live vaccine strain expressing the gp41 epitope at the surface of its flagellar filaments had higher titres of anti-peptide and anti-r-gp160 antibody and weak neutralizing activity on the IIIB isolate (90 % neutralization at 1 100 ). The sera of nine mice given two doses of the live vaccine by the oral route had either no or only very low titres of antibody to flagellar antigen d; they were therefore not tested for anti-peptide activity.

Neutralizing F(ab')2 fragments of protective monoclonal antibodies to yellow fever virus (YF) envelope protein fail to protect mice against lethal YF encephalitis.

Monoclonal antibodies (MAbs) prepared against the yellow fever virus (YF) vaccine strain 17D (17D YF) envelope E protein were used to investigate Fc piece involvement in antibody-mediated protection against YF encephalitis in mice 17D YF passaged either in Vero cells or in mouse brain (P-YF) to increase neurovirulence was used. To avoid uncertainty concerning antibody clearance and blood-brain barrier penetration, and to directly compare protective activity with neutralization in vitro, pre-formed antibody-virus complexes were injected intracerebrally or assayed for plaque formation in parallel. F(ab')2 fragments of an IgG2a MAb that strongly neutralized both YF strains retained molar equivalent neutralizing activity in vitro, but did not protect. However, further incubation of such F(ab')2-virus antibody complexes with rabbit IgG, but not F(ab')2 anti-mouse IgG resulted in protection. To unambiguously test for Fc piece involvement in this model we derived an IgG2a isotype switch variant from a protective IgG1 MAb-secreting hybridoma and prepared F(ab')2 fragments of the derivative. Intact and fragmented antibodies exhibited weak neutralizing activity. The variant antibody failed to protect against P-YF, but against considerably less neurovirulent 17D YF its protective capacity was 10-fold higher than that of its IgG1 parent. F(ab')2 fragments of the variant did not protect. Together, these results provide strong evidence of an in vivo protective function for the anti-virion antibody Fc piece and indicate that in vitro neutralizing activity as a predictor of antibody protective capacity is dependent on Fc piece integrity and isotype.

Subunit interaction in B19 parvovirus empty capsids.

B19 parvovirus is a small single-stranded DNA virus with a genome that encodes only two structural proteins, designated VP1 and VP2. 60 copies of the structural proteins assemble into the viral capsid, with approximately 95% VP2 and 5% VP1. Recombinant empty capsids composed of VP2 alone or of VP2 and VP1 self-assemble into particles that are morphologically indistinguishable from full virions. Empty capsids containing both VP2 and VP1 elicit a strong neutralizing antibody response when used to immunize rabbits. Capsids containing only VP2 are similarly antigenic but elicit only weak neutralizing activity. We performed fine structure epitope mapping by measuring the reactivity of antisera raised against capsids composed of VP2 and VP1 or VP2 alone against 85 overlapping peptides spanning the sequence of the two structural proteins. A profile of the antigenic difference between empty capsids with and without VP1 was produced from the resulting data. This profile divided the sequence of the structural proteins into four regions that correlated well with expected viral structures. Thus, the addition of a small number of VP1 residues altered the antigenicity of the entire capsid. The major area of enhanced antigenicity is homologous to the spike of canine parvovirus, an area known to contain both neutralizing and host-range determinants. Our data are consistent with a model in which the unique region of VP1 is necessary for the virus to assume its mature capsid conformation.