Neutralizing Capacity Of Antibodies Research Articles

The antibody response in the bovine mammary gland is influenced by the adjuvant and the site of subcutaneous vaccination

Intramammary infections in cattle resulting in mastitis have detrimental effects on cows’ well-being, lifespan and milk production. In the host defense against S. aureus mastitis antibodies are thought to play an important role. To explore potential ways to increase antibody titers in the bovine mammary gland the effects of various adjuvants on the magnitude, isotype, and neutralizing capacity of antibodies produced following subcutaneous vaccine administration at different immunization sites were analyzed. In this study, α-toxoid was used as a model antigen and formulated in three different alum-based adjuvants: Alum–Saponin, Alum–Oil, and Alum–Saponin–Oil. Vaccines were administered near the suspensory ligament of the udder or in the lateral triangular area of the neck. At both immunization sites, immunization with α-toxoid in Alum–Saponin–Oil resulted in higher specific antibody titers in milk and serum as compared with Alum–Oil and Alum–Saponin, without favoring an IgG1, IgG2, or IgA response. Furthermore, the neutralizing capacity of milk serum and serum following immunization near the udder and in the neck was higher when Alum–Saponin–Oil was used as adjuvant compared with Alum–Oil and Alum–Saponin. Prime immunizations near the udder effectively increased both antibody isotype titers and neutralization titers, while prime plus boost immunizations were required to induce similar effects following immunization in the neck. Results indicate that subcutaneous administration of an Alum–Saponin–Oil based vaccine near the udder could be further explored for the development of a one-shot vaccination strategy to efficiently increase intramammary antibody responses.

Recombinant lipidated dengue-3 envelope protein domain III stimulates broad immune responses in mice

The linkage of an immunogen with a toll-like receptor ligand has great potential to induce highly potent immune responses with the initial features of antigen-presenting cell activation. In the current study, we expressed recombinant dengue-3 envelope protein domain III (D3ED III) in lipidated form using an Escherichia coli-based system. The recombinant lipidated dengue-3 envelope protein domain III (LD3ED III) augments the expression levels of IL-12 family cytokines. LD3ED III-immunized mice enhance wide ranges of T cell responses as indicated by IFN-γ, IL-17, IL-21 production. Additionally, LD3ED III-immunized mice increase the frequencies of anti-D3ED III antibody producing cells. The boosted antibody titers cover various IgG isotypes, including IgG1, IgG2a, IgG2b, and IgG3. Importantly, LD3ED III-immunized mice induce neutralizing antibody capacity associated with a reduction of viremia levels after challenges. In contrast, mice that are immunized with D3ED III formulated with aluminum phosphate (D3ED III/Alum) only enhance Th2 responses and boost IgG1 antibody titers. Neither neutralizing antibody responses nor the inhibition of viremia levels after challenge is observed in mice that are immunized with D3ED III/Alum. These results suggest that LD3ED III can induce broad profiles of cellular and humoral immune responses.

Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S. aureus immune evasion proteins.

Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to combat S. aureus at the site of infection. Hence, in the present study, immunization via mucosal (intranasal; IN), intramuscular (triangle of the neck; IM), intramammary (IMM) and subcutaneous (suspensory ligament; SC) routes were analyzed for their effects on the quantity of the antibody responses in serum and milk as well as the neutralizing capacity of the antibodies within serum. The experimental vaccine comprised the recombinant S. aureus immune evasion proteins extracellular fibrinogen-binding protein (Efb) and the leukotoxin subunit LukM in an oil-in-water adjuvant combined with a hydrogel and alginate. The highest titer increases for both Efb and LukM specific IgG1 and IgG2 antibody levels in serum and milk were observed following SC/SC immunizations. Furthermore, the harmful effects of Efb and leukotoxin LukMF’ on host-defense were neutralized by serum antibodies in a route-dependent manner. SC/SC immunization resulted in a significant increase in the neutralizing capacity of serum antibodies towards Efb and LukMF’, shown by increased phagocytosis of S. aureus and increased viability of bovine leukocytes. Therefore, a SC immunization route should be considered when aiming to optimize humoral immunity against S. aureus mastitis in cattle.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0243-7) contains supplementary material, which is available to authorized users.

A method to quantitate the neutralizing capacity of anti-therapeutic protein antibodies in serum and their correlation to clinical impact

A robust, quantitative method for assessing the neutralizing capacity of anti-therapeutic protein antibodies was developed and tested using 4 analytical assay platforms typically used for detection of anti-drug neutralizing antibodies. The method described here utilized titration of increasing concentrations of therapeutic protein into serum containing anti-therapeutic protein antibodies, either positive control antibodies or clinical samples. Neutralizing capacities were calculated by determining the EC50 from the titration curves. The neutralizing capacity of purified anti therapeutic protein antibodies was expressed in terms of “μg of drug neutralized per μg of anti-TP antibody” present. In the case of serum originating from clinical study subjects, the neutralizing capacity of the samples was expressed as “μg of drug neutralized per mL of serum”. A relative shift in EC50 values was observed as the amount of serum or antibody was changed resulting in a proportional shift of the calculated neutralizing capacity. Application of this approach using different assay platforms was consistent providing evidence for its potential to be a useful approach to characterize, qualify and compare neutralizing positive control antibody preparations or clinical samples. Using this methodology, we were able to draw a clear correlation between the neutralizing capacity and the effect of these antibodies on a clinical pharmacodynamic (PD) marker. Determination of the neutralizing capacity of antibody positive samples from subjects in a clinical study indicated direct correlation of pharmacodynamic results with non-response, partial response and response to high, mid and low neutralizing capacities respectively.

The neutralizing capacity of antibodies elicited by parainfluenza virus infection of African Green Monkeys is dependent on complement

The African Green Monkey (AGM) model was used to analyze the role of complement in neutralization of parainfluenza virus. Parainfluenza virus 5 (PIV5) and human parainfluenza virus type 2 were effectively neutralized in vitro by naïve AGM sera, but neutralizing capacity was lost by heat-inactivation. The mechanism of neutralization involved formation of massive aggregates, with no evidence of virion lysis. Following inoculation of the respiratory tract with a PIV5 vector expressing HIV gp160, AGM produced high levels of serum and tracheal antibodies against gp120 and the viral F and HN proteins. However, in the absence of complement these anti-PIV5 antibodies had very poor neutralizing capacity. Virions showed extensive deposition of IgG and C1q with post- but not pre-immune sera. These results highlight the importance of complement in the initial antibody response to parainfluenza viruses, with implications for understanding infant immune responses and design of vaccine strategies for these pediatric pathogens.

Dynamics of evolutionary competition between budding and lytic viral release strategies.

In this paper, we consider the evolutionary competition between budding and lytic viral release strategies, using a delay differential equation model with distributed delay. When antibody is not established, the dynamics of competition depends on the respective basic reproductive ratios of the two viruses. If the basic reproductive ratio of budding virus is greater than that of lytic virus and one, budding virus can survive. When antibody is established for both strains but the neutralization capacities are the same for both strains, consequence of the competition also depends only on the basic reproductive ratios of the budding and lytic viruses. Using two concrete forms of the viral production functions, we are also able to conclude that budding virus will outcompete if the rates of viral production, death rates of infected cells and neutralizing capacities of the antibodies are the same for budding and lytic viruses. In this case, budding strategy would have an evolutionary advantage. However, if the antibody neutralization capacity for the budding virus is larger than that for the lytic virus, the lytic virus can outcompete the budding virus provided that its reproductive ratio is very high. An explicit threshold is derived.

Neutralizing antibodies against porcine circovirus type 2 in liquid pooled plasma contribute to the biosafety of commercially manufactured spray-dried porcine plasma1

Neutralizing antibodies (NA) inherently present in pooled plasma collected at commercial abattoirs may provide some protection against potential porcine circovirus type 2 (PCV2) infectivity of plasma. Moreover, these NA may also contribute to the biosafety of spray-dried porcine plasma (SDPP). The objective of the study was to characterize and quantify the PCV2 antibody neutralizing capacity in pooled liquid porcine plasma and SDPP samples collected from industrial spray-drying facilities located in the Southeast and Midwest regions of the United States and the Northeast region of Spain. In the United States, PCV2 NA was determined in 1 sample of pooled liquid plasma from commercial spray-drying plants in the Southeast and 1 from the Midwest region. Obtained results were compared with those of a plasma sample from a PCV2 vaccinated sow and 1 from a PCV2 antibody negative sow. In Spain, 15 pooled liquid porcine plasma samples and 10 SDPP samples were collected at a commercial spray-drying plant total and NA against PCV2 were determined. Results with pooled liquid porcine plasma from commercial spray-drying facilities in the United States indicated that NA titers against PCV2 in these samples (log2 8.33 ± 0.41 and 9.0 ± 0.0) were similar or greater than the plasma from the PCV2-vaccinated sow (log2 6.33 ± 0.41). The analysis of U.S. samples indicated that liquid plasma diluted to 1:256 (10(-2.41)) was able to neutralize between 100 to 200 PCV2 virus particles or about 4 logs10 median tissue culture infective dose (TCID50) per milliliter. Similarly, samples from the Spanish pooled liquid plasma and the SDPP samples indicated an increased amount of NA activity against PCV2. Specifically, a dilution of 10(-2.47 ± 0.33) of plasma was able to inactivate 100 PCV2 virus particles; therefore, the inactivation capacity of commercial liquid plasma was greater than 10(4) TCID50/mL. The calculated 90% reduction in infected cells because of NA in pooled plasma samples (log2 8.2 ± 0.38) was less (P < 0.05) than in its concentrate form of SDPP (mean, log2 10.2 ± 0.85). In conclusion, PCV2 NA contained in liquid pooled plasma from market pigs was detected at greater concentrations than that from a vaccinated sow and that after spray-drying biological neutralizing activity was conserved, which implies that the inherent NA in liquid plasma may have an important role in the biosafety of commercially produced SDPP.

Comparison of competitive ligand-binding assay and bioassay formats for the measurement of neutralizing antibodies to protein therapeutics

Administration of biological therapeutic proteins can lead to unwanted immunogenicity in recipients of these products. The assessment and characterization of such immune reactions can be helpful to better understand their clinical relevance and how they relate to patient safety and therefore, have become an integral part of a product development program for biological therapeutics. Testing for anti-drug antibodies (ADA) to biological/biotechnology-derived therapeutic proteins generally follows a tiered approach. Samples are initially screened for binding antibodies; presumptive positives are then confirmed in a confirmatory assay; subsequently, confirmed-positive samples may be further characterized by titration and with a neutralizing antibody (NAb) assay. Regulatory guidances on immunogenicity state that assessing the neutralizing capacity of antibodies should preferably be done using functional bioassays, while recognizing that competitive ligand-binding (CLB) assays may be substituted when neutralizing bioassays are inadequate or not feasible. This manuscript describes case studies from four companies in which CLB assays and functional bioassays were compared for their ability to detect neutralizing ADA against a variety of biotechnology-derived therapeutic proteins. Our findings indicate that CLB assays are comparable to bioassays for the detection of NAbs, in some cases offering better detection sensitivity, lower variability, and less matrix interference.

P11-16. Transfer of HIV-1 from Langerhans and interstitial dendritic cells to T lymphocytes: protection mediated by antibodies?

Background Langerhans (LC) and interstitial dendritic cells (IDC) present in mucosal tissues are among the first HIV-1 target cells. Infected DC have been shown to transfer the virus to nearby CD4 T lymphocytes. We have demonstrated that neutralizing antibodies (NAb) were able to inhibit the transfer of HIV-1 from monocyte-derived DC to CD4-T lymphocytes. In the context of HIV-1 transmission at mucosal site, we analysed the transfer of HIV-1 from LC and IDC to CD4-T lymphocytes, and determined the capacity of NAb to inhibit this transfer.

Study on the relationships between neutralizing antibody response against heterologous virus and disease progression of HIV type 1 B'/C infected individuals in China

Objective To study the relationships between neutralizing antibody response against heterologous virus and disease progression in Chinese HIV-1 B'/C infected individuals. Methods Plasmas from HIV-1-infected individuals, grouped as HIV chronically infected or AIDS according to CD4+ count and clinical symptom, were tested for neutralizing activity against the three HIV-1 isolates with very low homology in vitro. Six two-fold dilutions of each plasma sample (from 1/10 to 1/320) were tested against each virus from the panel. Giving a 50% reduction in p24Ag compared with normal human plasma control wells was defined as positive. The breadth of the cross-neutralizing response was defined based on the number of viruses that were effectively neutralized by any given patient-derived plasma sample. The magnitude of the crossneutralizing response was defined based on the average neutralizing titer against all heterologous viruses. Resuits We found that there revealed a significant difference between HIV chronically infected and AIDS group in the breaths and magnitudes of neutralizing heterologous virus. There was higher prevalence for the frequency of neutralizing heterologous virus in HIV chronically infected than AIDS. The results showed that there was positive correlation between the breadths and magnitudes of neutralizing response against heterologous virus and the plasma HIV RNA level in HIV chronically infected group, while not in AIDS group. There was no association between the breadth of the neutralizing responses against heterologous virus and CD4 T cell counts. Conclusion The capacity of neutralizing antibodies against heterologous virus varied among different disease stage. There were higher titers of neutralizing antibodies in HIV chronically infected than AIDS group. The loss of neutralizing antibodies in plasma from AIDS group appears to be associated with a narrowing of the antibody response during disease progression. These suggest that the presence of neutralizing antibodies against hetreologous virus was associated with disease progression. Key words: HIV-1; B'/C subtype; Neutralizing antibodies; Heterologous virus

Time Frames for Neutralization during the Human Immunodeficiency Virus Type 1 Entry Phase, as Monitored in Synchronously Infected Cell Cultures

Characterization of the neutralizing interaction between antibody and virus is hindered by the nonsynchronized progression of infection in cell cultures. Discrete steps of the viral entry sequence cannot be discerned, and thus, the mode of antibody-mediated interference with virus infectivity remains undefined. Here, we magnetically synchronize the motion and cell attachment of human immunodeficiency virus type 1 (HIV-1) to monitor the progression of neutralization, both in solution and following virus attachment to the cell. By simultaneous transfer of all viral particles from reaction solution with antibody to the cell-bound state, the precise rate of neutralization of cell-free virus could be determined for each antibody. HIV-1 neutralization by both monoclonal and polyclonal antibody preparations followed distinct pseudo-first-order kinetics. For all antibodies, cell types, and HIV-1 strains examined, postattachment interference served a major role in the neutralizing effect. To monitor the progression of postattachment interference, we synchronized the entry process at initiation and measured the escape of cell-bound virus from antibody. We found that different antibodies neutralized the virus over different time frames during the entry phase. Virus was observed to progress through a sequence of shifting sensitivities to different antibodies during entry, suggested here to correlate with the exposure time of the target epitope on receptor-activated viral envelope proteins. Thus, by monitoring the progression of HIV-1 entry under synchronized conditions, we identify a new and significant determinant of antibody neutralization capacity, namely, the time frames for neutralization during the course of the viral entry phase.

A new ELISA for determination of potency in snake antivenoms

A competitive ELISA for potency determination of bothropic equine antivenom was developed and compared to the conventional in vivo ED 50 assay, with the aim of partially substituting the in vivo assay in the monitoring of antivenom immunoglobulin levels. On this purpose, blood samples were taken at different times during and after the immunization protocol of the lot of horses used for production of snake antivenom at the Instituto de Higiene, Uruguay. Both the competitive ELISA and the ED 50 assay were performed on those samples. In addition, a group of five commercial pepsin-digested antivenoms were tested by both methods. A significant ( P<0.001) correlation (Pearson's r=0.957) was found between the ELISA titres and the corresponding ED 50 values, indicating that the in vitro test can estimate the neutralizing antibody capacity of the sera as well as the in vivo assay. By means of this new ELISA, it was found that the immunized animals maintained good venom antibody titres, in the order of 20–50% of the maximum achieved, even 10 month after the end of the immunization schedule. The main advantage of our ELISA design is its ability to correctly estimate the neutralization capacity of crude hyperimmune plasma and antivenom sera independently of their antibody composition in terms of whole IgG or F(ab′) 2 fragment.

Antibody response after RSV infection in children younger than 1 year of age living in a rural area of Mozambique.

Serological responses have been studied in respiratory syncytial virus (RSV) infected children < 1 year of age attending the outpatient department of the Manhiça District Hospital (Mozambique). Molecular characterization of viral RNA in nasopharyngeal aspirates from the infected children indicated a high level of genetic uniformity among the infecting viruses, all of which belonged to a single genotype of RSV group A. A representative virus strain, Moz00, was isolated from one of the infants and was used, together with the group A strain A2 and the group B strain 8/60, as antigens in the quantification of infant antibody responses. In this study, 97.5% (39/40) and 96.4% (27/28) of infected children produced an antibody response against Moz00 detected by the membrane fluorescent antibody test (MFAT) and the neutralization test (NT), respectively. Seroconversion rates decreased when the A2 and 8/60 strains were used as antigen in MFAT (95.4% and 88.2%, respectively) or NT (81.8% and 54.5%, respectively), indicating that antibody responses had both group- and strain-specific components. Antibodies in convalescent sera of infected children were compared with maternally derived antibodies detected in a group of children also < 1 year of age, but with no evidence of RSV infection. The convalescent sera exhibited reduced neutralizing capacity when the 8/60 strain was used as antigen (P = 0.028), suggesting that the infant antibody response lacks neutralizing capacity against strains of the heterologous virus group. Restricted cross-reactivity and neutralizing capacity of antibodies generated by young children might be expected to induce only moderate protection in subsequent epidemics against genetically distant strains.

Antibody responses to the repetitive Plasmodium falciparum antigen Pf332 in humans naturally primed to the parasite.

Antibodies to the degenerate repeats of EB200, a part of the Plasmodium falciparum antigen Pf332, are protective in monkeys. To analyse the prevalence, magnitude and specificity of antibodies to EB200 in malaria-exposed humans, the IgG antibody reactivity with recombinant EB200 protein as well as with crude malaria antigen was determined in Senegalese donors (n = 100; 4-87 years). Antibody reactivity with EB200 was low or absent in children below 15 years but was prevalent and significantly higher in older donors. In comparison, all individuals displayed reactivity with a crude malaria antigen preparation, which also increased with age. The reactivity with the crude malaria antigen was correlated to the reactivity with EB200, suggesting that the low levels of IgG to EB200 found in some adult donors reflected a limited degree of recent exposure to parasites rather than a selective non-responsiveness to Pf332. Comparison of serological and clinical data showed that high levels of antibodies to crude malaria antigen and to EB200 were predictive of fewer future clinical attacks of malaria. A reactivity pattern very similar to that found in Senegalese donors was observed in Liberian adults where 80% of the sera showed reactivity with EB200 and all peptides were recognized by between 60 and 100% of the donors. This strong reactivity with EB200-derived overlapping peptides suggests that the epitopes in EB200, to a large extent, are linear. In the light of previous data on the parasite neutralizing capacity of antibodies to Pf332, the present results emphasize the potential interest of Pf332-derived sequences for inclusion in a subunit vaccine against P. falciparum malaria.

Diagnostic Approach of Phospholipid-Dependent Antibodies

Recent scientific developments enable to better understand the strong heterogeneity of assay methods for phospholipid (PLP)-dependent antibodies, measured as lupus anticoagulant (LA) or as anti-PLP antibodies (APA). These latter are actually targeted at complexes of β₂-glycoprotein I (β2GPI) and anionic PLP and also react with insolubilized β2GPI alone in some patients. Although APA present little species specificity for β2GPI, some of them bind preferentially to complexes of human β2GPI and PLP. LAs are diagnosed with screening assays (dPT, dRVVT, KCT, APTT); their sensitivity is dependent on the concentration and type of PLPs used. They are either β2GPI or prothrombin dependent. Based on the antibody-neutralizing capacity of PLP preparations (such as hexagonal phase phosphatidylethanolamine) or their bypassing activity, confirmatory assays enable to confirm LA. LAs and APAs bind to different epitopes on β2GPI or its complexes with PLP. Major characteristics of APA assays are: the capture antigen (PLPs, β2GPI source); its optimization and density on micro-ELISA plates; the efficacy of postcoating saturation; the animal serum used for saturation and diluent; the second antibody which must avoid nonspecific interactions; the calibrators proposed and their reference to international standards; the cutoff between the normal and the pathological ranges; the assay sensitivity and overall specificity for APAs. New optimized assays have been developed to meet these criteria. They are designed with PLP-coated plates, saturated first with immunoglobulin-free human serum as a source of β2GPI, then with goat serum also used in diluent. The cutoff value is determined with at least 200 normal plasma samples (mean + 3 standard deviations or 99th percentiles) and plasma samples from patients without APAs. This approach offers both optimized specificity and sensitivity for testing APAs, along with a good standardization and it helps to measure the true APAs associated with pathology.

Antigenic domain 1 of human cytomegalovirus glycoprotein B induces a multitude of different antibodies which, when combined, results in incomplete virus neutralization.

Glycoprotein B (gB, gpUL55) is the major antigen for the induction of neutralizing antibodies against human cytomegalovirus (HCMV), making it an attractive molecule for active and passive immunoprophylaxis. The region between aa 552 and 635 of HCMV gB (termed AD-1) has been identified as the immunodominant target for the humoral immune response following natural infection. AD-1 represents a complex domain which requires a minimal continuous sequence of more than 70 aa for antibody binding. Neutralizing as well as non-neutralizing antibodies can bind to AD-1 in a competitive fashion. The fine specificity of AD-1-binding monoclonal antibodies (MAbs) and affinity-purified human polyclonal antibodies was analysed by using recombinant proteins containing single amino acid substitutions spanning the entire AD-1 domain. Our results revealed that all MAbs had individual patterns of binding to the mutant proteins indicating the presence of a considerable number of distinct antibody-binding sites on AD-1. The neutralization capacity of antibodies could not be predicted from their binding pattern to AD-1 mutant proteins. Polyclonal human antibodies purified from different convalescent sera showed identical binding patterns to the mutant proteins suggesting that the combined antibody specificities present in human sera are comparable between individuals. Neutralization capacities of polyclonal human AD-1 antibodies did not exceed 50% indicating that, during natural infection, a considerable proportion of non-neutralizing antibodies are induced and thus might provide an effective mechanism to evade complete virus neutralization.

Specificity and neutralizing capacity of antibodies elicited by non-toxic protein (TsNTxP) of the scorpion Tityus serrulatus

Specificity and neutralizing capacity of antibodies elicited by non-toxic protein (TsNTxP) of the scorpion Tityus serrulatus

Analysis of Viral Antigens Using Biosensor Technology

The different types of epitopes found in viral antigens are described. The BIAcore has been used successfully to map the location of epitopes in viral antigens and several examples of such studies are presented. Knowledge of the affinity of viral antibodies is important for understanding the molecular basis of viral antigenicity and the mechanism of infectivity neutralization by antibodies. The use of the BIAcore for measuring binding constants of viral antibodies is described. Affinity constants of antibodies binding to virus particles are easily obtained using sensor chips on which the virus is immobilized by a first layer of antibodies. Affinity constants obtained with the BIAcore agree closely with values obtained by several other methods of affinity determination. By comparing the binding properties of neutralizing and nonneutralizing viral antibodies with the BIAcore, it should be possible to establish whether there is a relationship between affinity and neutralizing capacity of antibodies. By immobilizing synthetic peptides corresponding to viral epitopes on sensor chips, the BIAcore can also be used to quantitate antibody to specific epitopes present in polyclonal antiserum.

Influence of carbohydrate moieties on the immunogenicity of human immunodeficiency virus type 1 recombinant gp160.

The role of carbohydrates in the immunogenicity of human immunodeficiency virus type 1 (HIV-1) glycoproteins (gp160 and gp120) remains poorly understood. We have analyzed the specificity and neutralizing capacity of antibodies raised against native gp160 or against gp160 deglycosylated by either endo F-N glycanase, neuraminidase, or alpha-mannosidase. Rabbits immunized with these immunogens produced antibodies that recognized recombinant gp160 (rgp160) from HIV-1 in a radioimmunoassay and in an enzyme-linked immunosorbent assay. Antibodies elicited by the different forms of deglycosylated gp160 were analyzed for their reactivity against a panel of synthetic peptides. Compared with anti-native gp160 antisera, serum reactivity to most peptides remained unchanged, or it could increase (peptide P41) or decrease. Only antibodies raised against mannosidase-treated gp160 failed to react with a synthetic peptide (peptide P29) within the V3 loop of gp120. Rabbits immunized with desialylated rgp160 generated antibodies which recognized not only rgp160 from HIV-1 but also rgp140 from HIV-2 at high titers. Although all antisera produced against glycosylated or deglycosylated rgp160 could prevent HIV-1 binding to CD4-positive cells in vitro, only antibodies raised against native or desialylated gp160 neutralized HIV-1 infectivity and inhibited syncytium formation between HIV-1-infected cells and noninfected CD4-positive cells, whereas antibodies raised against alpha-mannosidase-treated gp160 inhibited neither virus replication nor syncytium formation. These findings indicate that the carbohydrate moieties of gp160 can modulate the specificity and the protective efficiency of the antibody response to the molecule.

Specificity and neutralizing capacity of antibodies elicited by a synthetic peptide of scorpion toxin.

Polyclonal antibodies raised against a synthetic peptide (sequence 50-59) of Androctonus australis Hector toxin II can neutralize the effects of toxin II in vivo. The antigenic specificities of anti-peptide and anti-toxin antibodies were compared by competitive aqueous phase radioimmunoassay by using 125I-toxin II, chemically modified or homologous toxins, and the synthetic peptide 50-59, either free or bound to bovine serum albumin (BSA). The antipeptide and anti-toxin antibodies had a comparable high affinity for the native toxin, but anti-peptide antibodies exhibited a lower binding capacity. Anti-peptide antibodies had a higher affinity for native toxin than for the peptide 50-59 bound to BSA, used as immunogen, and were unable to recognize the free peptide. These results suggest that it is necessary to restrict the conformational freedom of the immunizing peptide in order to obtain anti-peptide antibodies with a high affinity for the toxin. The lysine residue at position 58 of toxin II, essential for toxicity, appears to be immunogenic when immunization is with peptide 50-59 bound to BSA and not with the native toxin. This residue is antigenic in the native toxin, however, as shown by the anti-peptide antibodies.