Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins

Summary.  A chimera of the two immunodominant African swine fever (ASF) virus proteins p54 and p30 was constructed by insertion of the gene CP204L into a Not I restriction site of E183L gene. The resulting chimeric protein p54/30, expressed by a recombinant baculovirus in insect cells and in Trichoplusia ni larvae, retained antigenic determinants present in both proteins and reacted in Western blot with a collection of sera from inapparent ASF virus carrier pigs. Remarkably, pigs immunized with the chimeric protein developed neutralizing antibodies and survived the challenge with a virulent African swine fever virus, presenting a reduction of about two logs in maximum viremia titers with respect to control pigs. In conclusion, this study revealed that the constructed chimeric protein may have utility as a serological diagnostic reagent and for further immunological studies that may provide new insights on mechanisms of protective immunity to ASFV. Accepted May 18, 2001 Received March 29, 2001     

Recombinant Antigen Targets for Serodiagnosis of African Swine Fever

African swine fever (ASF) is an infectious and economically important disease of domestic pigs. There is no vaccine, and so reliable diagnosis is essential for control strategies. The performance of four recombinant ASF virus (ASFV) protein (pK205R, pB602L, p104R, and p54)-based enzyme-linked immunosorbent assays (ELISAs) was evaluated with European porcine field sera that had been established by Office International des Epizooties (OIE)-approved tests to be ASFV negative (n = 119) and ASFV positive (n = 80). The κ values showed that there was almost perfect agreement between the results of the “gold standard” test (immunoblotting) and the results obtained by the p54-specific ELISA (κ = 0.95; 95% confidence interval [CI], 0.90 to 0.99) and the pK205R-specific ELISA or the pB602L-specific ELISA (κ = 0.92; 95% CI, 0.86 to 0.97). For the pA104R-specific ELISA, there was substantial to almost perfect agreement (κ = 0.81; 95% CI, 0.72 to 0.89). Similar results were observed by the OIE-approved ELISA (κ = 0.89; 95% CI, 0.82 to 0.95). Importantly, antibodies against these proteins were detectable early after infection of domestic pigs. Preliminary testing of 9 positive and 17 negative serum samples from pigs from West Africa showed identical results by the recombinant protein-based ELISA and the OIE-approved tests. In contrast, there was a high degree of specificity but a surprisingly a low level of sensitivity with 7 positive and 342 negative serum samples from pigs from East Africa. With poorly preserved sera, only the p104R-specific ELISA showed a significant reduction in sensitivity compared to that of the OIE-approved ELISA. Finally, these recombinant proteins also detected antibodies in the sera of the majority of infected warthogs. Thus, recombinant ASFV proteins p54, pB602L, and pK205R provide sensitive and specific targets for the detection of antibodies in European and West African domestic pigs and warthogs.African swine fever (ASF) virus (ASFV) is an icosahedral cytoplasmic DNA virus that infects pigs and soft ticks of the Ornithodoros genus. This virus is the sole member of the family Asfarviridae (6). ASFV has variable pathogenicity in domestic pigs, with infections ranging from being highly lethal to subclinical. Infection of wildlife mammalian hosts, the warthog and the bushpig, on the other hand, results in an unapparent, nonpathogenic infection, which provides a potentially dangerous reservoir of virus. There is no vaccine. Therefore, rapid and specific diagnostic procedures are an essential component of any control strategy. In addition, the presence of virus strains with reduced virulence and the resulting presence of asymptomatic infected animals (4, 11) make the serological diagnosis the only realistic basis for the control of the disease in affected countries. As a general rule, pigs that survive natural infection develop antibodies against ASFV from 7 to 10 days after infection. These antibodies persist for long periods of time (16), perhaps due to continuous antigenic stimulation by the frequent occurrence of persistent infection. Thus, antibody detection is a rational approach to the detection of the subacute and chronic forms of the disease.The role of specific antibodies in immunity to ASFV infection in pigs has been controversial. The passive transfer of anti-ASFV antibodies delays the onset of clinical signs but does not consistently protect animals from eventual death (25, 26, 17). Similarly, vaccination with the putative protective proteins p30 and p54 conferred protection to only 50% of the tested animals (10). In a different study, in which no protection was observed after immunization against p54, p30, and p72, the only effects detected were a delay in the onset of clinical disease and a reduction in the level of viremia (15). Such observations emphasize the role of cell-mediated immune responses during ASFV infection. Indeed, a positive correlation was observed between the stimulation of NK cell activity and the absence of clinical symptoms after experimental infection, suggesting that NK cells play an important role in protective immunity (13). In addition to NK cells, CD8⁺ T cells may also play a role, as their depletion in vivo abrogates protective immunity to ASFV infection (18). Therefore, immunity to ASFV is likely to be due to a combination of both serological and cellular mechanisms. This complexity of the porcine immune response to ASFV has impaired the development of an effective vaccine but does justify diagnosis on the basis of the detection of antibodies.Current Office International des Epizooties (OIE)-approved assays for ASFV-specific antibody determination consist of an initial screening of sera by enzyme-linked immunosorbent assay (OIE-ELISA), followed by an immunoblotting assay to confirm the results for samples with doubtful and positive results. These OIE-approved tests are based on the use of live virus as the antigen and involve the requirement of level 3 biosafety facilities for the production and handling of the pathogen (16, 20, 21). The risk associated with the handling of live virus, together with the lack of reliability of the OIE-ELISA for the analysis of poorly preserved samples so often encountered in sera of African origin (1, 3), provides the stimulus for the development of alternative and more robust systems for the detection of anti-ASFV antibodies. Indeed, previous studies have demonstrated that recombinant viral proteins can give improved specificity and sensitivity when they are applied to the analysis of European field sera (9, 19, 22).In previous studies, 12 serological immunodeterminants of ASFV were characterized by exhaustive screening of a representative lambda phage cDNA expression library of the tissue culture-adapted Ba71V isolate of ASFV for antibodies (12). These included four proteins encoded by previously unassigned open reading frames (ORFs) (B602L, C44L, CP312R, and K205R), as well as some of the more well studied structural proteins (pA104R, p10, p32, p54, and p73) and three enzymes (RNA reductase, DNA ligase, and thymidine kinase). The complete sequence of each of these proteins was then recloned into pGEX for expression in Escherichia coli, followed by purification of the recombinant proteins and testing against sera from experimentally infected animals. Four of these proteins (p54/E183L, histone-like/pA104R, pB602L, and pK205R) were revealed to be promising targets for both immunoglobulin G (IgG) and IgM antibody responses (23), and further validation of these proteins as targets is the focus of this work. The results obtained by the analysis of a large collection of serum samples from susceptible animals from Europe and Africa were comparable to the results obtained by the OIE-ELISA prescribed for use for international trade.     

Enhanced discrimination of African swine fever virus isolates through nucleotide sequencing of the p54, p72, and pB602L (CVR) genes

Complete sequencing of p54-gene from 67 European, American, and West and East African Swine Fever virus (ASFV) isolates revealed that West African and European ASFV isolates classified within the predominant Genotype I according to partial sequencing of p72 were discriminated into four major sub-types on the basis of their p54 sequences. This highlighted the value of p54 gene sequencing as an additional, intermediate-resolution, molecular epidemiological tool for typing of ASFV viruses. We further evaluated p54-based genotyping, in combination with partial sequences of two other genes, for determining the genetic relationships and origin of viruses responsible for disease outbreaks in Kenya. Animals from Western and central Kenya were confirmed as being infected with ASFV using a p72 gene-based PCR assay, following outbreaks of severe hemorrhagic disease in domestic pigs in 2006 and 2007. Eleven hemadsorbing viruses were isolated in macrophage culture and genotyped using a combination of full-length p54-gene sequencing, partial p72-gene sequencing, and analysis of tetrameric amino acid repeat regions within the variable region of the B602L gene (CVR). The data revealed that these isolates were identical in their p72 and p54 sequence to viruses responsible for ASF outbreaks in Uganda in 2003. There was a minor difference in the number of tetrameric repeats within the B602L sequence of the Kenyan isolates that caused the second Kenyan outbreak in 2007. A practical implication of the genetic similarity of the Kenyan and Ugandan viral isolates is that ASF control requires a regional approach.     

The structural protein p54 is essential for African swine fever virus viability

Protein p54, one of the most antigenic structural African swine fever virus (ASFV) proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. Attempts to inactivate the p54 gene from ASFV by targeted insertion of β-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. To demonstrate that, we inserted in the TK (thymidine kinase) locus of the virus a construction containing a second copy of the p54 gene and β-glucuronidase selection marker under the control of p54 and p73 promoters, respectively. Virus mutant clones expressing a second copy of p54 and β-glucuronidase were used to achieve deletion mutants of the original copy of the gene. Virus mutants expressing only the second inserted copy of p54 and the two selection markers mentioned above were successfully obtained. Therefore, we have demonstrated that the p54 gene product plays an essential role in virus growth, characterizing for the first time in ASFV an essential virus gene.     

Strong sequence conservation of African swine fever virus p72 protein provides the molecular basis for its antigenic stability

Summary The major capsid protein p72 of African swine fever virus (ASFV) has long been considered an important immunodominant antigen for serologic diagnosis. Here we describe the cloning and sequence analysis of two p72-coding genes from ASFV strains Uganda (UGA) and Dominican Republic-2 (DR2). Sequence comparison of these genes, together with those from two other ASFV strains (BA71V and E70), demonstrated that the p72 proteins are highly conserved (97.8% to 100% amino acid sequence identity) in strains isolated from different parts of the world. These results support previous observations indicating that p72 is antigenically stable, and provide a useful molecular basis for further development of ASFV serologic tests using this important antigenic molecule.     

Optimization and validation of recombinant serological tests for African Swine Fever diagnosis based on detection of the p30 protein produced in Trichoplusia ni larvae

We describe the validation of an enzyme-linked immunosorbent assay (ELISA) and confirmatory immunoblotting assays based on a recombinant p30 protein (p30r) produced in insect larvae using a baculovirus vector. Such validation included the following: (i) the scaling up and standardization of p30r production and the associated immunoassays, (ii) a broad immunological analysis using a large number of samples (a total of 672) from Spain and different African locations, and (iii) the detection of the ASF virus (ASFV)-antibody responses at different times after experimental infection. Yields of p30r reached up to 15% of the total protein recovered from the infected larvae at 3 days postinfection. Serological analysis of samples collected in Spain revealed that the p30r-based ELISA presented similar sensitivity to and higher specificity than the conventional Office International des Epizooties-approved ASFV ELISA. Moreover, the p30r ELISA was more sensitive than the conventional ELISA test in detecting ASFV-specific antibodies in experimentally infected animals at early times postinfection. Both the recombinant and conventional ELISAs presented variable rates of sensitivity and specificity with African samples, apparently related to their geographical origin. Comparative analyses performed on the sequences, predicted structures, and antigenicities of p30 proteins from different Spanish and African isolates suggested that variability among isolates might correlate with changes in antigenicity, thus affecting detection by the p30r ELISA. Our estimations indicate that more than 40,000 ELISA determinations and 2,000 confirmatory immunoblotting tests can be performed with the p30r protein obtained from a single infected larva, making this a feasible and inexpensive strategy for production of serological tests with application in developing countries.     

Modulation of splenic macrophages,and swine leukocyte antigen (SLA) and viral antigen expression following African swine fever virus (ASFV) inoculation

Summary Expression of viral and major histocompatibility complex (MHC) antigens and localization of T cells and macrophages was studied in frozen tissue sections of spleens taken from normal pigs or from pigs inoculated with highly virulent Lisbon 60 (L60), or with moderately virulent Dominican Republic 1978 (DR-II), African swine fever virus (ASFV) isolates. Splenic sections from L60 inoculated pigs exhibited a large decrease in macrophage staining, whereas DR-II infected animals appeared more intensely stained in the macrophage sheath arteries. Class I and class II MHC expression was decreased in spleens from pigs infected with either isolate at 3 day post inoculation (DPI). This was reversed in DR-II inoculated pigs at 4 DPI. Splenic tissue sections from L60 inoculated pigs exhibited only a marginal increase in SLA expression at a later time, 6 DPI. We suggest that the recovery of SLA expression during infection of pigs with ASFV is associated with survival or replacement of macrophages in the spleen leading to an effective immune response against the virus.     

Enhanced immunity and protective efficacy against SIVmac251 intrarectal challenge following ad-SIV priming by multiple mucosal routes and gp120 boosting in MPL-SE

Previously, 39% of rhesus macaques primed orally, intranasally, and intratracheally with adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinants and boosted with gp120 in monophosphoryl lipid A-stable emulsion (MPL-SE) remained aviremic or cleared or controlled viremia at the threshold of detection following SIV(mac251) intrarectal challenge (Study B). In contrast, no macaques primed orally and intranasally with Ad-SIV recombinants and boosted with gp120 in Quillaja Saponaria-21 exhibited undetectable viremia post-challenge (Study A). We conducted a detailed comparison of the studies to elucidate the effect of different vaccine regimens on induced immunity associated with the different challenge outcomes. Quantitative viral load comparisons were statistically analyzed. All immune responses were assessed at identical timepoints post-immunization, and cellular immunity was re-evaluated on cryopreserved cells from Study B macaques to match Study A data acquired with frozen cells. Study B exhibited greater protective efficacy, increased levels of p11C and p54m tetramer positive cells and a trend toward enhanced interferon-gamma secreting cells in response to Env and Gag peptides, modestly enhanced serum neutralizing antibodies, and greater positivity in anti-gp120 rectal IgA and IgG antibodies. Study A macaques exhibited greater positivity in salivary IgA anti-gp120 antibodies. Thus, the vaccine regimen using oral-intranasal-intratracheal priming and protein boosting in MPL-SE was superior, eliciting greater protective efficacy against pathogenic SIV(mac251) and enhanced SIV-specific immunity, systemically and at rectal sites. The mechanism(s) by which binding antibodies, lacking neutralizing activity against the primary challenge virus, may contribute to protection requires further study.     

The immunological response of pigs and Guinea pigs to antigens of African swine fever virus

Summary Pigs vaccinated with glutaraldehyde-fixed alveolar macrophages (AM) infected with African swine fever virus (ASFV) had an accelerated serological response after subsequent challenge and a slight reduction in levels of viraemia. Vaccination of pigs with detergent-treated infected AM produced no detectable serological response and no protection against homologous challenge.Guinea pigs were vaccinated with glutaraldehyde-fixed ASFV-infected cells, detergent-treated infected cells, detergent-treated infected spleen homogenate, purified ASFV or sonicated infected cells. Antibody was detectable by ELISA after vaccination with all preparations except the detergent-treated infected spleen vaccine. However, vaccination with purified ASFV or sonicated infected cells induced antibodies that were also strongly reactive in antibody-dependent cell-mediated cytotoxicity and complement-mediated lysis assays. If such antibodies are protective, immunization of pigs with purified ASFV or sonicated infected cells may induce a protective immunity.     

Antibodies neutralizing feline leukaemia virus (FeLV) in cats immunized with the transmembrane envelope protein p15E

The feline leukaemia virus (FeLV) vaccines that are currently in wide use are generally poor inducers of virus-neutralizing antibodies, although such antibodies appear after recovering from challenge. However, the presence of neutralizing antibodies in cats recovering from natural FeLV infection clearly correlates with resistance to subsequent infection and passive transfer of antibodies can protect other animals. After demonstrating the induction of neutralizing antibodies in rats and goats immunized with the transmembrane envelope protein p15E of FeLV, cats were immunized with the same antigen. High titres of neutralizing antibodies specific for FeLV were induced and epitope mapping revealed a pattern of recognition similar to that seen following immunization of rats and goats. These epitopes are highly related to epitopes recognized after immunization with porcine endogenous retrovirus (PERV) p15E and to epitopes recognized by neutralizing antibodies in patients infected with human immunodeficiency virus type 1. The ability of p15E to induce neutralizing antibodies in cats suggests that it should be included in the next generation of vaccines. In contrast, sera from FeLV-infected animals usually fail to recognize the neutralization-relevant epitopes in p15E. Since homologous epitope sequences are present in feline endogenous retroviruses, it appears that tolerance against these sequences is not induced.     

Immunization with recombinant protein: conditions for cytotoxic T cell and/or antibody induction

Safe vaccines should optimally induce both cell-mediated and humoral immunity. Recently, it has been shown that protective cytotoxic T cells (CTLs) can be induced not only with live vaccines, but also with recombinant viral proteins. This report shows in C57BL/6 (H-2^b) mice that the recombinant nucleoprotein (N) of vesicular stomatitis virus (VSV) induced protective CTLs but no neutralizing antibodies in mice, whereas the recombinant glycoprotein (G) of VSV alone induced neutralizing antibodies but no CTLs. If the N and G of VSV were coinjected, both CTLs and a long-lasting neutralizing IgG response was measurable, demonstrating that mixed vaccines can be used to induce protective CTLs and antibodies with an efficiency comparable to live virus. In an attempt to define optimal conditions for CTL priming, the intravenous, intraperitoneal and subcutanous route of injection were compared. Intravenous injection of recombinant VSV-N induced up to 30 times higher responses than the latter two routes. Finally, we tried to define conditions inducing only CTLs and no antibodies binding to the native protein form, or vice versa, only antibodies and no CTLs. Intravenous injection of boiled VSV-N induced a CTL response but no antibodies specific for the native VSV-N, whereas VSV-N injected subcutanously in incomplete Freund's adjuvant induced high amounts of anti-VSV-N antibodies but virtually no CTLs. The conditions defined here permit vaccines to be designed which would function along selected and defined immunological effector pathways.     

Aujeszky's disease vaccination and infection of pigs with maternal immunity: Effects on cell- and antibody-mediated immunity

Summary SCC, ADV-SCC, ADV-ADCC and ADV-LYST as well as ND₅₀-titres of neutralizing serum antibodies were examined in 36 passively immune pigs, 25 of which were vaccinated at 3 weeks of age and partly revaccinated 3 weeks later. Twenty-five vaccinated animals and 8 non-immune control pigs were challenged with infectious ADV.Independent of the state of maternal immunity the cytotoxic response of the white blood cells from all the animals was low at WPP 3 but rose with increasing age. ADV-LYST occurred only in some of the animals. A single vaccination evoked no significant effect on our immune parameters, but revaccination led to higher ADV-LYST and ADV-ADCC. In pigs vaccinated at WPP3 the neutralizing serum titres decreased gradually, similar to unvaccinated animals, indicating that the antibodies were of maternal origin. However, after vaccination at WPP6, no further decline of ND₅₀-titres could be detected, pointing to a limited antibody production. Animals vaccinated at WPP3 and revaccinated 3 weeks later showed a significant increase of serum neutralizing titres.Whereas the controls showed typical symptoms of Aujeszky's disease, the immune animals, especially the unvaccinated passively immune pigs, showed only elevated temperatures and most of them excreted small amounts of ADV.The development of cellular immunity after infection was rather similar within the maternally immune group independent whether the animals had been vaccinated or not, but ADV-ADCC and ADV-LYST showed a more rapid progress within the vaccinated group than in the non-vaccinated group and the non-immune control group. Infection resulted in significantly higher ND₅₀ titres in vaccinated and revaccinated animals than in unvaccinated animals, indicating a secondary response in those pigs. Thus, ADV sensitization of lymphocytes had been evoked by vaccination despite the presence of maternal antibody.The interpretation of the results was complicated by great individual and litter-dependent variations of the immune parameters.Abbreviations ADV Aujeszky's disease virus - ADV-ADCC antibody-dependent cell-mediated cytotoxicity against ADV infected cells - ADV-LYST lymphocyte stimulation by ADV - ADV-SCC spontaneous cell-mediated cytotoxicity against ADV infected cells - CTI cytotoxic index(ices) - DPI day post infection - LYST lymphocyte stimulation - SCC spontaneous cell-mediated cytotoxicity - SI stimulation index(ices) - WPP week post partum - WPV week post vaccination - WPRV week post revaccination With 5 Figures     

Multiplication and distribution of Aujeszky's disease (pseudorabies) virus in vaccinated and non-vaccinated pigs after intranasal infection

Summary The primary sites of Aujeszky's disease virus (ADV) multiplication in intranasally (i.n.) infected pigs were found to be in the nasopharyngeal, tracheal and pulmonary regions. From the second day post infection (DPI) onward ADV invaded the central nervous system and other organs. The virus was isolated from the nasopharyngeal region for at least 2 weeks. In serum ADV was present with low levels from DPI 1 to DPI 7.In pigs vaccinated with an inactivated vaccine and then challenged the distribution of ADV was rather similar to that in non-vaccinated animals, in spite of the presence of neutralizing antibodies. The virus titres in the organs generally were lower than in non-vaccinated animals up to DPI 7. Thereafter, titre differences were no longer significant. Virus was isolated from the tonsils and the lungs for at least 2 weeks. Interferon production in vaccinated infected pigs was significantly lower than in non-vaccinated infected pigs. Though multiplication and dissemination of ADV occurred, vaccinated pigs did not show clinical symptoms of Aujeszky's disease.Traces of ADV were detected in a small percentage of white blood cells (WBC) of non-vaccinated infected pigs. ADV was isolated from the lymphocyte-enriched and polymorphnuclear leukocyte-enriched fractions, but not from the monocyte-enriched fractions, apparently on account of the small cell number. Multiplication of ADV was demonstrated in cultured WBC from some of the vaccinated and non-vaccinated infected animals.The results are discussed with regard to neural spread of ADV and the role of haematogenic or lymphatic dissemination of the virus by WBC and to humoral and cell-mediated immunity in vaccinated pigs.Abbrevations ADV Aujeszky's disease virus - CNS central nervous system - DPI day(s) post infection - FCS fetal calf serum - i.n. intranasal(ly) - Ly lymphocyte(s) - MEM Eagle's minimal essential medium - Mono monocyte(s) - PMNL polymorphonuclear leukocyte(s) - WBC white blood cells (buffy coat) With 1 Figure     

Some investigations on the adjuvant mechanism of DEAE dextran

In vitro it was shown that adsorption of inactivated FMDV onto DEAE-D kieselgur columns did not occur in the presence of 0.1--0.15M NaCl. These NaCl concentrations are present in DEAE-D/FMDV vaccines and in the tissues of animals. Therefore, adsorption of virus antigen does not appear to be responsible for the adjuvant effect of DEAE-D. In pigs it was demonstrated that DEAE-D exerts its optimal adjuvant effect, as measured by the formation of neutralizing antibodies and protection against challenge infection, when injected together with inactivated FMDV as vaccine. Apart from this, a good adjuvant effect (group immunity 75--100 per cent) was evoked in about one half and a moderate effect (group immunity 50--70 per cent) in about a quarter of the inoculated animals even if DEAE-D was separately injected locally and temporally from the inactivated virus. With regard to immunity it apparently does not matter whether DEAE-D or inactivated virus was given first, but an interval of 48 hours or 4 days between injection seemed to be more favourable than one of 24 hours. With regard to the formation of neutralizing antibodies the situation is comparable to that of immunity with the exception that a time interval of 24 hours between the application of DEAE-D and inactivated virus or vice versa was as good as that of 48 hours or 4 days. The results are discussed in regard to the possible mechanism of the adjuvant effect of DEAE-D on the cellular level.     

Role of Humoral Immunity in Host Defense Against HIV

Individuals infected with HIV-1 and nearly everyone vaccinated with HIV-1 vaccines will, in time, generate antibodies against viral proteins. These antibodies do not resolve natural infection, and vaccine candidates that successfully stimulate the production of high titers of neutralizing antibodies have failed to protect against infection. In spite of this, antibodies continue to be a focus of vaccine research. One reason for the continued interest in antibodies is the failure of a vaccine engineered to generate cell-mediated immunity against HIV. Successful protective immunity against most intracellular pathogens involves several arms of the immune response. A successful vaccine should also stimulate both protective cell-mediated immunity and specific antibody. Efforts should be directed toward making a vaccine that will stimulate the production of 1) more antibody, 2) more broadly cross-reactive neutralizing antibody (broadly neutralizing antibodies), and 3) antibody with a particular functional activity (antibody-dependent cell-mediated cytotoxicity; catalytic antibodies).     

Antibodies and B cell memory in viral immunity

Humoral immunity, in particular secreted neutralizing antibodies, is of central importance to protect the body against acutely cytopathic viruses, whereas noncytopathic viruses have found ways of balanced coexistence with the immune system to avoid antibody-mediated elimination. There is evidence that polyspecific "natural" antibodies provide early protection, independent of T cell help. If that line of defense is crossed, T cell-dependent immune responses then generate a humoral memory provided by long-lived plasma cells secreting specific antibodies of adapted avidity and function, i.e., isotype, even in the absence of virus. Secreted protective antibodies of humoral memory provide an efficient line of defense against reinfection and are backed up by specific B and T memory cells of reactive memory. Whereas humoral memory has developed effective antiviral protection, some viruses (i.e., HIV) have managed to develop specific evasion strategies to escape it. Thus, coevolution provides us with some insight into just how substantial antiviral antibodies and memory B cell are in protecting the host from virus infection.     

Characterization of epitopes for neutralizing monoclonal antibodies to classical swine fever virus E2 and Erns using phage-displayed random peptide library

Summary. Infection of cells with classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoproteins E2 and E^rns with receptor molecules on the cell surface. These proteins are also the major antigens for eliciting neutralizing antibodies and conferring protective immunity. Here we report the identification of multiple neutralizing epitopes on these proteins by screening a phage-displayed random peptide library with CSFV-specific neutralizing monoclonal antibodies. Two different E2-specific neutralizing mAbs (a18 and 24/10) were found to bind to a common motif SPTxL, which is similar to the sequence SPTTL of the E2 protein (aa 289–293), indicating that this is likely to be an immunodominant epitope. Similarly, an immunodominant epitope corresponding to the sequence DKN of E^rns (aa 117–119) was identified for two independent E^rns-specific neutralizing antibodies, b4-22 and 24/16, respectively. Another binding motif, CxNNxTC, was identified for mAb 24/16, but not for b4-22. Sequencing analysis of the genes coding for the light chain of these mAbs was conducted to ensure that all mAbs were derived from different hybridomas, rather than from different subclones of a common parent line. Inhibition studies using immunofluorescent antibody assay and virus neutralization test demonstrated that the mimotope peptides truly mimicked the antibody binding determinants on the viral proteins. The detailed mapping data for these neutralizing epitopes will be useful for development of improved diagnostic tests and perhaps a peptide-based vaccine for this important swine disease.     

Structure-based,targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition

The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization.