Antibody Avidity Maturation Research Articles

Analysis of antibodies avidity for Tityus serrulatus scorpion venom in antivenom production and its potential for application as a potency test

Antivenoms are the only specific medication for neutralizing toxins present in venom of animals such scorpions and snakes through antigen-antibody binding. Several analyses are carried out throughout its production in order to ensure the quality and effectiveness of the antivenom that will be administered to the patient. One of these is the potency assay, which is performed to assess the ability of antivenoms to neutralize the toxic effects of the venom injected in mice. The substitution of in vivo for in vitro assays such as ELISA has been presented by other authors, bringing several advantages such as the reduction in the use of animals, in costs and in the duration of the assays. However, the avidity index of antivenom antibodies determined by ELISA has not yet been applied for this purpose. Therefore, the objective of this study was to evaluate the avidity of sera from hyperimmunized horses with crude Tityus serrulatus venom, a scorpion species associated with the most serious accidents in Brazil, and its potential for application as a potency test replacing the in vivo assay. The avidity ELISA proved to be interesting for monitoring the binding strength of antibodies produced by horses in hyperimmune plasma production programs. It was possible to verify oscillations in antibody avidity that occurred along the immunization cycles, differences between novice and veteran horses, maturation of antibody avidity, and correlation between avidity index and antibody titre. Similar results were obtained for crude venom and purified Ts1 toxin. In addition, the avidity ELISA apparently demonstrated potential for application as a potency test in the initial stage of antivenom production. However, more studies are necessary.

RBD and Spike DNA-Based Immunization in Rabbits Elicited IgG Avidity Maturation and High Neutralizing Antibody Responses against SARS-CoV-2.

Neutralizing antibodies (nAbs) are a critical part of coronavirus disease 2019 (COVID-19) research as they are used to gain insight into the immune response to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections. Among the technologies available for generating nAbs, DNA-based immunization methods are an alternative to conventional protocols. In this pilot study, we investigated whether DNA-based immunization by needle injection in rabbits was a viable approach to produce a functional antibody response. We demonstrated that three doses of DNA plasmid carrying the gene encoding the full-length spike protein (S) or the receptor binding domain (RBD) of SARS-CoV-2 induced a time-dependent increase in IgG antibody avidity maturation. Moreover, the IgG antibodies displayed high cross neutralization by live SARS-CoV-2 and pseudoviruses neutralization assays. Thus, we established a simple, low cost and feasible DNA-based immunization protocol in rabbits that elicited high IgG avidity maturation and nAbs production against SARS-CoV-2, highlighting the importance of DNA-based platforms for developing new immunization strategies against SARS-CoV-2 and future emerging epidemics.

Antibody Avidity Maturation Following Recovery From Infection or the Booster Vaccination Grants Breadth of SARS-CoV-2 Neutralizing Capacity.

Cross-neutralizing capacity of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is important in mitigating (re-)exposures. Role of antibody maturation, the process whereby selection of higher affinity antibodies augments host immunity, to determine SARS-CoV-2 neutralizing capacity was investigated. Sera from SARS-CoV-2 convalescents at 2, 6, or 10 months postrecovery, and BNT162b2 vaccine recipients at 3 or 25 weeks postvaccination, were analyzed. Anti-spike IgG avidity was measured in urea-treated ELISAs. Neutralizing capacity was assessed by surrogate neutralization assays. Fold change between variant and wild-type neutralization inferred the breadth of neutralizing capacity. Compared with early-convalescent, avidity indices of late-convalescent sera were significantly higher (median, 37.7 [interquartile range 28.4-45.1] vs 64.9 [57.5-71.5], P < .0001). Urea-resistant, high-avidity IgG best predicted neutralizing capacity (Spearman r = 0.49 vs 0.67 [wild-type]; 0.18-0.52 vs 0.48-0.83 [variants]). Higher-avidity convalescent sera better cross-neutralized SARS-CoV-2 variants (P < .001 [Alpha]; P < .01 [Delta and Omicron]). Vaccinees only experienced meaningful avidity maturation following the booster dose, exhibiting rather limited cross-neutralizing capacity at week 25. Avidity maturation was progressive beyond acute recovery from infection, or became apparent after the booster vaccine dose, granting broader anti-SARS-CoV-2 neutralizing capacity. Understanding the maturation kinetics of the 2 building blocks of anti-SARS-CoV-2 humoral immunity is crucial.

Antibody avidity, persistence, and response to antigen recall: comparison of vaccine adjuvants

Differences in innate immune ‘imprinting’ between vaccine adjuvants may mediate dissimilar effects on the quantity/quality of persisting adaptive responses. We compared antibody avidity maturation, antibody/memory B cell/CD4+ T cell response durability, and recall responses to non-adjuvanted fractional-dose antigen administered 1-year post-immunization (Day [D]360), between hepatitis B vaccines containing Adjuvant System (AS)01B, AS01E, AS03, AS04, or Alum (NCT00805389). Both the antibody and B cell levels ranked similarly (AS01B/E/AS03 > AS04 > Alum) at peak response, at D360, and following their increases post-antigen recall (D390). Proportions of high-avidity antibodies increased post-dose 2 across all groups and persisted at D360, but avidity maturation appeared to be more strongly promoted by AS vs. Alum. Post-antigen recall, frequencies of subjects with high-avidity antibodies increased only markedly in the AS groups. Among the AS, total antibody responses were lowest for AS04. However, proportions of high-avidity antibodies were similar between groups, suggesting that MPL in AS04 contributes to avidity maturation. Specific combinations of immunoenhancers in the AS, regardless of their individual nature, increase antibody persistence and avidity maturation.

Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Avidity Maturation and Association with Disease Severity.

The kinetics of IgG avidity maturation during SARS-CoV-2 infection was studied. The IgG avidity assay used a novel label-free immunoassay technology. It was found that there was a strong correlation between IgG avidity and days since symptom onset, and peak readings were significantly higher in severe than mild disease cases.

Brief Report: Selection of HIV-1 Variants With Higher Transmission Potential by 1% Tenofovir Gel Microbicide.

Women in the CAPRISA 004 trial assigned to use 1% tenofovir (TFV) microbicide gel, who became HIV-1 infected, had higher viral load set-point and slower antibody avidity maturation compared with placebo participants. We investigated whether TFV gel was selected for viruses with altered genetic characteristics. The participants of the CAPRISA 004 trial (n = 28 TFV and 43 placebo) were from KwaZulu-Natal Province, South Africa and were infected with HIV-1 subtype C. After HIV-1 diagnosis, they were recruited into the CAPRISA 002 cohort. We analyzed gag sequences from the earliest time point post infection (within 3 months of estimated time of infection). Transmission index was measured using a model which predicts the likelihood of an amino acid to be transmitted. Phylogenetic distance from a regional consensus sequence was calculated from a maximum likelihood phylogenetic tree. Transmission index and distance from the most common (consensus) sequence have been shown to be markers of transmission fitness. We found that viruses infecting TFV gel recipients were closer to the consensus sequence of regional strains (P = 0.003) and had higher transmission index (P = 0.01). The transmission index was weakly correlated with concomitant viral load (Spearman r = 0.22, P = 0.06). Decreased acquisition risk may have increased the barrier to infection therefore selecting for fitter, more consensus-like viruses. Such virus fitness effects will need to be considered for future pre-exposure prophylaxis and vaccine trials.

Persistence and avidity maturation of antibodies to A(H1N1)pdm09 in healthcare workers following repeated annual vaccinations

Healthcare workers are at increased risk of influenza infection through direct patient care, particularly during the early stages of a pandemic. Although influenza vaccination is widely recommended in Healthcare workers, data on long-term immunogenicity of vaccination in healthcare workers are lacking.The present study was designed to assess the persistence of the humoral response after pandemic vaccination as well as the impact of repeated annual vaccination in healthcare workers (n=24).Pandemic influenza vaccination resulted in a significant increase in haemagglutination inhibition (HI) antibody titers with 93–100% of subjects achieving protective titers 21-days post each of the three annual vaccinations. Seroprotective antibodies measured by HI, microneutralization and single radial hemolysis assays were present in 77–94% of healthcare workers 6 months post-vaccination. Repeated vaccination resulted in an increased duration of seroprotective antibodies with seroprotective titers increasing from 35–62% 12 months after 2009 pandemic vaccination to 50–75% 12 months after 2010 vaccination. Furthermore, repeated annual vaccination augmented the avidity of influenza-specific IgG antibodies.In conclusion, we have shown that A(H1N1)pdm09 vaccination induces high seroprotective titers that persist for at least 6 months. We demonstrate that repeated vaccination is beneficial to healthcare workers and results in further avidity maturation of vaccine-induced antibodies.

Antibody Maturation in Women Who Acquire HIV Infection While Using Antiretroviral Preexposure Prophylaxis.

The CAPRISA 004 preexposure prophylaxis (PrEP) randomized trial demonstrated that women who used a vaginal gel containing the antiretroviral drug tenofovir (TFV) had a 39% lower risk of acquiring human immunodeficiency virus (HIV). It is not known whether topical TFV alters the antibody response to breakthrough HIV infection. In this study, antibody maturation was evaluated using 3 serologic assays: the BED capture enzyme immunoassay (CEIA), the Bio-Plex (Luminex) assay, and the Bio-Rad avidity assay. Tests were performed using serum samples collected 3, 6, 9, 12, 24, 36, 48, and >48 months after seroconversion from 95 women in the CAPRISA 004 trial (35 in the TFV gel arm and 60 in the placebo arm). For the BED CEIA and Luminex assay, linear mixed effects models were used to examine test results by study arm. Cox proportional hazard analysis was used to examine time to avidity cutoff. Anti-HIV antibody titers did not differ between study arms. Women assigned to TFV gel demonstrated slower antibody avidity maturation, as determined by the Bio-Rad (P = .04) and gp120 Bio-Plex (P = .028) assays. Women who were assigned to receive topical TFV but became infected had slower antibody avidity maturation, with potential implications for diagnosis and antibody-based incidence assays as access to antiretroviral therapy-based PrEP is increased.

Conjugate-like immunogens produced as protein capsular matrix vaccines

Capsular polysaccharides are the primary antigenic components involved in protective immunity against encapsulated bacterial pathogens. Although immunization of adolescents and adults with polysaccharide antigens has reduced pathogen disease burden, pure polysaccharide vaccines have proved ineffective at conferring protective immunity to infants and the elderly, age cohorts that are deficient in their adaptive immune responses to such antigens. However, T-cell-independent polysaccharide antigens can be converted into more potent immunogens by chemically coupling to a "carrier protein" antigen. Such "conjugate vaccines" efficiently induce antibody avidity maturation, isotype switching, and immunological memory in immunized neonates. These immune responses have been attributed to T-cell recognition of peptides derived from the coupled carrier protein. The covalent attachment of polysaccharide antigens to the carrier protein is thought to be imperative to the immunological properties of conjugate vaccines. Here we provide evidence that covalent attachment to carrier proteins is not required for conversion of T-independent antigens into T-dependent immunogens. Simple entrapment of polysaccharides or a d-amino acid polymer antigen in a cross-linked protein matrix was shown to be sufficient to produce potent immunogens that possess the key characteristics of conventional conjugate vaccines. The versatility and ease of manufacture of these antigen preparations, termed protein capsular matrix vaccines (PCMVs), will likely provide improvements in the manufacture of vaccines designed to protect against encapsulated microorganisms. This in turn could improve the availability of such vaccines to the developing world, which has shown only a limited capacity to afford the cost of conventional conjugate vaccines.

Plasmodium falciparum merozoite surface protein 2: epitope mapping and fine specificity of human antibody response against non-polymorphic domains.

BackgroundTwo long synthetic peptides representing the dimorphic and constant C-terminal domains of the two allelic families of Plasmodium falciparum merozoite surface proteins 2 are considered promising malaria vaccine candidates. The aim of the current study is to characterize the immune response (epitope mapping) in naturally exposed individuals and relate immune responses to the risk of clinical malaria.MethodsTo optimize their construction, the fine specificity of human serum antibodies from donors of different age, sex and living in four distinct endemic regions was determined in ELISA by using overlapping 20 mer peptides covering the two domains. Immune purified antibodies were used in Western blot and immunofluorescence assay to recognize native parasite derivate proteins.ResultsImmunodominant epitopes were characterized, and their distribution was similar irrespective of geographic origin, age group and gender. Acquisition of a 3D7 family and constant region-specific immune response and antibody avidity maturation occur early in life while a longer period is needed for the corresponding FC27 family response. In addition, the antibody response to individual epitopes within the 3D7 family-specific region contributes to protection from malaria infection with different statistical weight. It is also illustrated that affinity-purified antibodies against the dimorphic or constant regions recognized homologous and heterologous parasites in immunofluorescence and homologous and heterologous MSP2 and other polypeptides in Western blot.ConclusionData from this current study may contribute to a development of MSP2 vaccine candidates based on conserved and dimorphic regions thus bypassing the complexity of vaccine development related to the polymorphism of full-length MSP2.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-510) contains supplementary material, which is available to authorized users.

The MSHA Strain of Pseudomonas Aeruginosa Activated TLR Pathway and Enhanced HIV-1 DNA Vaccine Immunoreactivity

The mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA) has been shown to trigger naïve immune responses through the activation of monocytes, macrophages, natural killer cells (NK cells) and antigen presenting cells (APCs). Based on the hypothesis that PA-MSHA activates natural immunity through the Toll-like receptor (TLR) pathway, we scanned several critical TLR pathway molecules in mouse splenocytes using high-throughput real-time QRT-PCR and co-stimulatory molecule in bone marrow-derived dendritic cells (BMDCs) following in vitro stimulation by PA-MSHA. PA-MSHA enabled activation of the TLR pathway mediated by NF-κB and JNK signaling in splenocytes, and the co-stimulatory molecule CD86 was up-regulated in BMDCs. We then assessed the adjuvant effect of PA-MSHA for HIV-1 DNA vaccines. In comparison to DNA inoculation alone, co-inoculation with low dosage of PA-MSHA enhanced specific immunoreactivity against HIV-1 Env in both cellular and humoral responses, and promoted antibody avidity maturation. However, high doses of adjuvant resulted in an immunosuppressive effect; a two- or three-inoculation regimen yielded low antibody responses and the two-inoculation regimen exhibited only a slight cellular immunity response. To our knowledge, this is the first report demonstrating the utility of PA-MSHA as an adjuvant to a DNA vaccine. Further research is needed to investigate the exact mechanisms through which PA-MSHA achieves its adjuvant effects on innate immune responses, especially on dendritic cells.

Delayed Maturation of Antibody Avidity but Not Seroconversion in Rhesus Macaques Infected With Simian HIV During Oral Pre-Exposure Prophylaxis

Pre-exposure prophylaxis (PrEP) is a novel intervention strategy for the prevention HIV transmission. Because several clinical trials are at various stages of completion, it is important to understand the impact of PrEP treatment on the development of the immune response to HIV, particularly in individuals who exhibit breakthrough infections despite PrEP. A model of HIV infection, using rhesus macaques and the simian/human immunodeficiency virus (SHIV), was used to evaluate the effects of PrEP on the evolution of the humoral immune response. Time to seroconversion, neutralizing and binding antibody levels, and antibody avidity were measured in 12 rhesus macaques infected during daily or intermittent PrEP with FTC (emtricitabine) or Truvada (FTC/tenofovir combination) and compared with 11 untreated, simian HIV-infected controls. Macaques that became infected while receiving PrEP exhibited significantly lower peak virus loads during acute infection as compared with untreated animals. Although the timing of seroconversion and SHIV binding and neutralizing antibody levels were not impacted by treatment, lower maturation rates of antibody avidity for anti-p27, gp120, gp160, and gp41 were observed. This study suggests that reduced virus loads associated with PrEP treatment have little impact on timing of seroconversion and neutralizing/binding antibody levels; however, maturation of antibody avidity was suppressed.

Fusion of C3d molecule with neutralization epitope(s) of hepatitis E virus enhances antibody avidity maturation and neutralizing activity following DNA immunization

Previous studies have identified that a hepatits E virus peptide (HEV-p179), spanning amino acids (aa) 439–617 in the 660-aa protein encoded by open reading frame 2(ORF2) of the Chinese epidemic strain (genotype 4), is the minimal size fragment of conformation-dependent neutralization epitope(s). We report here the successful immunization of mice with DNA vaccines expressing the secreted form of HEV-p179 (fused with a human tissue plasminogen activator (tPA) signal sequence) and the tPA-p179-C3d fusion protein (fused with three tandem copies of the murine complement C3d). Analysis of antibody responses in vaccinated mice revealed that immunizations with tPA-p179-C3d3 DNA vaccine dramatically increased both the level and avidity maturation of antibodies against HEV-p179 compared to p179 and tPA-p179 DNA vaccines. In addition, this increased antibody response correlated with neutralizing titers in a PCR-based cell culture neutralization assay. These results indicate that vaccination with C3d conjugated p179 DNA vaccine enhances antibody responses to HEV, and this approach may be applied to overcome the poor immunogenicity of DNA vaccines to generate HEV neutralizing antibodies.

Enhancement of Antibody Responses toBacillus anthracisProtective Antigen Domain IV by Use of Calreticulin as a Chimeric Molecular Adjuvant

The generation of protective humoral immune responses against the receptor-binding domain (domain IV) of protective antigen [PA(dIV)] of Bacillus anthracis represents a plausible approach against anthrax toxin. In the current study, we have developed a naked DNA vaccine encoding calreticulin (CRT) linked to PA(dIV) of Bacillus anthracis [CRT/PA(dIV)]. We transfected a human embryonic kidney cell line (HEK 293) with CRT/PA(dIV) DNA and performed Western blotting and confocal microscopy analysis. We found that linkage of CRT to PA(dIV) targets PA(dIV) to the endoplasmic reticulum, resulting in secretion of the chimeric CRT/PA(dIV) protein. We then evaluated the ability of CRT/PA(dIV) DNA to generate PA(dIV)-specific antibody responses and protective immunity against lethal anthrax toxin (PA plus lethal factor) challenge. We found that mice immunized with CRT/PA(dIV) DNA were capable of rapidly inducing significantly higher PA(dIV)-specific antibody responses than mice immunized with PA(dIV) DNA alone. Furthermore, we observed that this enhanced antibody response generated by CRT/PA(dIV) DNA was CD4 dependent, since CD4 knockout mice demonstrated a significant reduction in antibody responses. In addition, analysis of the titers and avidity maturation of the induced PA-specific antibodies revealed that vaccination with CRT/PA(dIV) DNA vaccine accelerated the avidity maturation of antibodies to PA(dIV) compared to vaccination with PA(dIV) DNA. Importantly, the enhanced antibody responses correlated to protective immunity against lethal anthrax toxin challenge. Thus, DNA vaccines encoding CRT linked to PA(dIV) may dramatically enhance PA-specific protective antibody responses. Our results have significant clinical applications for biodefense against anthrax toxin.

Effective Highly Active Antiretroviral Therapy in Patients With Primary HIV-1 Infection Prevents the Evolution of the Avidity of HIV-1-Specific Antibodies

To evaluate if the administration of highly active antiretroviral therapy (HAART) during primary HIV infection (PHI) may affect the antibody avidity evolution. In 13 subjects with symptomatic PHI, of whom 8 initiated HAART at diagnosis, the Avidity Index (AI) and Western blot evolution patterns were analyzed on serial serum/plasma samples for 1 year. In 4 patients, who subsequently interrupted HAART, additional specimens were analyzed. At diagnosis, the range of HIV viremia was 0.003 to 38 x 10(6) copies/mL. In untreated patients, viremia reached the set point in 4 to 6 months, whereas in treated patients, early suppression of viremia was observed, remaining undetectable during therapy. At diagnosis, the median AI was low in untreated (0.42, range: 0.33 to 0.43) and treated (0.44, range: 0.40 to 0.72) patients. At 3, 6, and 12 months, the AI progressively increased in untreated patients, whereas it remained <0.80 in all treated patients. In the 4 patients interrupting HAART, the AI increased after therapy interruption to greater than 0.80 in < or = 6 months. The Western blot pattern transiently/partially reversed during HAART in 2 patients. Antibody avidity maturation takes place only in the presence of ongoing viral replication. These results may have relevant implications in understanding the complex mechanism of maturation of the immune response to HIV.

Human Immunodeficiency Virus (HIV) Antibody Avidity Testing To Identify Recent Infection in Newly Diagnosed HIV Type 1 (HIV-1)-Seropositive Persons Infected with Diverse HIV-1 Subtypes

A guanidine-based antibody avidity assay for the identification of recently acquired human immunodeficiency virus type 1 (HIV-1) infection was evaluated. The kinetics of maturation of antibody avidity were determined prospectively in 23 persons undergoing acute seroconversion followed for up to 1,075 days. Avidity indices (AI) of <or=0.75 and <or=0.80 reproducibly identified seroconversion within the previous 125 (95% confidence interval [CI], 85 to 164) and 142 (95% CI, 101 to 183) days, respectively. To validate the assay, 432 serum samples from newly diagnosed patients were tested by both the avidity assay and the detuned assay. Results were highly concordant (kappa value for agreement, 0.85). The avidity assay was subsequently used to screen 134 consecutive newly diagnosed patients, including 55/134 (41%) infected with non-B subtypes (A, C, D, G, CRF01, CRF02, CRF06, CRF13, and CRF16). In this cohort, 25/79 (32%) persons with the B subtype and 7/55 (13%) with non-B subtypes showed an AI of <or=0.75, and there were 16/25 (64%) and 3/7 (43%) persons, respectively, with a documented history of acute seroconversion illness within the predicted seroconversion interval. An AI of <or=0.75 was also observed for four patients (three with the B subtype and one with a non-B subtype) who presented with AIDS-defining conditions. In multivariate analysis, an AI of <or=0.75 was associated with younger age, higher HIV-1 plasma RNA load, and being born in the United Kingdom or Ireland rather than in Africa but not with gender, ethnicity, risk group, HIV-1 subtype, or CD4 counts. In conclusion, HIV antibody avidity testing provides a reliable method for identifying recently acquired HIV-1 infection. Results are affected by advanced disease and should therefore be interpreted in the context of other clinical parameters.

Combined Conjugate Vaccines: Enhanced Immunogenicity with the N19 Polyepitope as a Carrier Protein

The N19 polyepitope, consisting of a sequential string of universal human CD4(+)-T-cell epitopes, was tested as a carrier protein in a formulation of combined glycoconjugate vaccines containing the capsular polysaccharides (PSs) of Neisseria meningitidis serogroups A, C, W-135, and Y. Good antibody responses to all four polysaccharides were induced by one single immunization of mice with N19-based conjugates. Two immunizations with N19 conjugates elicited anti-MenACWY antibody titers comparable to those induced after three doses of glycoconjugates containing CRM197 as carrier protein. Compared to cross-reacting material (CRM)-based constructs, lower amounts of N19-MenACWY conjugates still induced high bactericidal titers to all four PSs. Moreover, N19-MenACWY-conjugated constructs induced faster and higher antibody avidity maturation against meningococcal C PS than CRM-based conjugates. Very importantly, N19-specific antibodies did not cross-react with the parent protein from which N19 epitopes were derived, e.g., tetanus toxoid and influenza virus hemagglutinin. Finally, T helper epitopes of the N19 carrier protein were effectively generated both in vivo (after immunization with the N19 itself) and in vitro (after restimulation of epitope-specific spleen cells). Taken together, these data show that the N19 polyepitope represents a strong and valid option for the generation of improved or new combined glycoconjugate vaccines.

Antibody Avidity Maturation during Severe Acute Respiratory Syndrome–Associated Coronavirus Infection

The maturation of virus-specific immunoglobulin G avidity during severe acute respiratory syndrome–associated coronavirus infection was examined. The avidity indices were low (mean ± SD, 30.8% ± 11.6%) among serum samples collected ⩽50 days after fever onset, intermediate (mean ± SD, 52.1% ± 14.1%) among samples collected between days 51 and 90, and high (mean ± SD, 78.1% ± 8.0%) among samples collected after day 90. Avidity indices of 40% and 55% could be considered as cutoff values for determination of recent (⩽50 days) and past (>65 days) infection, respectively. Measurement of antibody avidity can be used to differentiate primary infection from reexposure and to assess humoral responses to candidate vaccines

Development of Functional Haemophilus Influenzae Type B Antibodies after Vaccination of Autologous Stem Cell Transplant Recipients.

The conditioning regiments for autologous stem cell transplantation (aSCT) lead to impairment of the immune system and susceptibility to infections. Although infectious complications are more prominent in allogeneic transplantation, it is also a major source of morbidity in patients undergoing aSCT. Therefore, vaccination of patients after stem cell transplantation has been recommended. We conducted a prospective follow-up study to determine quantitative and qualitative aspects of the humoral immune response to multiple vaccinations with conjugated Haemophilus influenzae type b (Hib) vaccine in autologous stem cell transplant recipients. Sixteen patients (twelve with multiple myeloma and four with non-Hodgkin's lymphoma) received a conjugated Hib vaccine at 6, 8 and 14 months after transplantation. Antibody titres were determined by ELISA, 21 days after each vaccination. Adequate anti-Hib antibody responses (i.e. a four fold or greater increase in antibody levels in addition to a minimal titre of 50 U/ml corresponding to 18.8 μg/ml) were achieved in 7 (44%) of the patients after one vaccination. A second and third vaccination resulted in an adequate anti-Hib antibody response in 12 (75%) and 14 (88%) patients, respectively. Functionality of anti-Hib antibodies was assessed by measurement of antibody avidity. Repeated vaccination induced maturation of antibody avidity as demonstrated by increased resistance to sodium thiocyanate (NaSCN) treatment (Figure 1; the avidity index (AI) is defined as the molarity of NaSCN at which 50% of the amount of IgG antibodies bound to the coated antigen in the absence of NaSCN has been eluded. Mean AI plus range are shown after one, two and three vaccinations.). Subsequently, opsonizing activity of antibodies was determined by a phagocytosis assay. FITC-labelled Hib was incubated with serum and added to fresh polymorphonuclear cells (PMN). Mean fluorescence intensity (MFI) was measured by flowcytometry. Anti-Hib antibodies supported phagocytosis by PMN's after multiple vaccinations (Figure 2; MFI at different time points is shown). In conclusion, multiple vaccinations with Hib-conjugate vaccine in autologous stem cell recipients result in high antibody response rates and functional maturation of antibodies. [Display omitted] [Display omitted]

PorA-specific differences in antibody avidity after vaccination with a hexavalent Men B outer membrane vesicle vaccine in toddlers and school children

A clinical phase II trial with an experimental hexavalent outer membrane vesicle (OMV) vaccine (HexaMen) containing six different porin A (PorAs) was carried out in toddlers (2–3 years) and schoolchildren (7–8 years) in The Netherlands. HexaMen exists of two OMVs each containing three different PorA types. The serum bactericidal activity (SBA) after vaccination against the six PorAs was significantly different and was higher in toddlers than in schoolchildren. After vaccination the SBA against P1.5-2,10 was 4–6 times higher than against P1.7–2,4. The aim of this study was to test whether the differences in SBA could be explained by a difference in subtype-specific antibody avidity maturation. The avidity index (AI) of antibodies against three subtypes (PorA types P1.5-2,10; P1.12-1,13 and P1.7-2,4) was measured by ELISA and evaluated in relation to SBA. A significant avidity maturation for the 3 PorA subtypes was found. This maturation was most pronounced for P1.5-2,10 (mean AI=72%), correlating with the highest SBA titres. Generally, the avidity titre correlated best with SBA. No differences in avidity indices against the three tested PorAs were found between toddlers and school children indicating that avidity maturation induced by this vaccine is not age-dependent.