Broad Antibody Responses Research Articles

Factors Associated with the Development of Cross-Reactive Neutralizing Antibodies during Human Immunodeficiency Virus Type 1 Infection

The characterization of the cross-reactive, or heterologous, neutralizing antibody responses developed during human immunodeficiency virus type 1 (HIV-1) infection and the identification of factors associated with their generation are relevant to the development of an HIV vaccine. We report that in healthy HIV-positive, antiretroviral-naïve subjects, the breadth of plasma heterologous neutralizing antibody responses correlates with the time since infection, plasma viremia levels, and the binding avidity of anti-Env antibodies. Anti-CD4-binding site antibodies are responsible for the exceptionally broad cross-neutralizing antibody responses recorded only in rare plasma samples. However, in most cases examined, antibodies to the variable regions and to the CD4-binding site of Env modestly contributed in defining the overall breadth of these responses. Plasmas with broad cross-neutralizing antibody responses were identified that targeted the gp120 subunit, but their precise epitopes mapped outside the variable regions and the CD4-binding site. Finally, although several plasmas were identified with cross-neutralizing antibody responses that were not directed against gp120, only one plasma with a moderate breadth of heterologous neutralizing antibody responses contained cross-reactive neutralizing antibodies against the 4E10 epitope, which is within the gp41 transmembrane subunit. Overall, our study indicates that more than one pathway leads to the development of broad cross-reactive neutralizing antibodies during HIV infection and that the virus continuously escapes their action.

Frequency and phenotype of human immunodeficiency virus envelope-specific B cells from patients with broadly cross-neutralizing antibodies.

Induction of broadly cross-reactive neutralizing antibodies (NAb) is an important goal for a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. Some HIV-infected patients make a NAb response that reacts with diverse strains of HIV-1, but most candidate vaccines have induced NAb only against a subset of highly sensitive isolates. To better understand the nature of broad NAb responses that arise during natural infection, we screened patients for sera able to neutralize diverse HIV strains and explored the frequency and phenotype of their peripheral Envelope-specific B cells. We screened 113 HIV-infected patients of various clinical statuses for the prevalence of broad NAb. Sera able to neutralize at least four of five viral isolates were found in over one-third of progressors and slow progressors, but much less frequently in aviremic long-term nonprogressors. Most Env-specific antibody-secreting B cells were CD27(hi) CD38(hi) plasmablasts, and the total plasmablast frequency was higher in HIV-infected patients than in uninfected donors. We found that 0.0031% of B cells and 0.047% of plasmablasts secreted Env-specific immunoglobulin G (IgG) in an enzyme-linked immunospot (ELISPOT) assay. We developed a novel staining protocol to label HIV-specific B cells with Env gp140 protein. A total of 0.09% of B cells were found to be Env-specific by this method, a frequency far higher than that indicated by ELISPOT assay. gp140-labeled B cells were predominantly CD27(+) and surface IgG(+). These data describe the breadth and titer of serum NAb and the frequency and phenotype of HIV-specific B cells in a cohort of patients with broad cross-neutralizing antibody responses that are potential goals for vaccines for HIV.

Characterization of the humoral immune response to islet antigen 2 in children with newly diagnosed type 1 diabetes.

To characterize the humoral immune response to islet antigen 2 (IA-2) in patients with newly diagnosed type 1 diabetes (T1D), we compared the profile of epitope- and isotype-specific IA-2 antibodies (IA-2A) between children with a humoral immune response restricted to IA-2 and children with a broad response including insulin autoantibodies (IAA) and antibodies to glutamic acid decarboxylase (GADA) in addition to IA-2A. The study subjects (n=100) were derived from a consecutive series of 1108 patients from the Finnish Pediatric Diabetes Register (investigators listed in the Appendix). Islet cell antibodies, IAA, GADA, total IA-2A levels, IA-2/IA-2beta epitopes, and isotypes were measured, and human leukocyte antigen (HLA) genotypes were analyzed. There were no significant differences between the two groups in the frequency or levels of epitope-specific IA-2A. Those with an IA-2-restrictive response tested positive more frequently for IgA-IA-2A (P=0.001), had higher titers of IgE-IA-2A (P=0.025), tested positive for more IA-2A isotypes than the broad responders (P=0.04), and carried the high-risk HLA-(DR4)-DQB1*0302 haplotype more frequently than those with a broad antibody response (P=0.019). These data show that children with newly diagnosed T1D, who test positive only for IA-2A out of the three molecular antibodies predictive of T1D, have a broader IA-2-specific isotype response and stronger association with the high-risk HLA haplotype than those testing positive for all three molecular antibodies. This may be indicative of a different pathogenetic mechanism in those with their humoral immune response restricted to IA-2 at the time of diagnosis.

Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses

Although approximately 3 % of the world's population is infected with Hepatitis C virus (HCV), there is no prophylactic vaccine available. This study reports the design, cloning and purification of a single polyprotein comprising the HCV core protein and non-structural proteins NS3, NS4a, NS4b, NS5a and NS5b. The immunogenicity of this polyprotein, which was formulated in alum, oil-in-water emulsion MF59 or poly(dl-lactide co-glycolide) in the presence or absence of CpG adjuvant, was then determined in a murine model for induction of B- and T-cell responses. The addition of adjuvants or a delivery system to the HCV polyprotein enhanced serum antibody and T-cell proliferative responses, as well as IFN-gamma responses, by CD4+ T cells. The antibody responses were mainly against the NS3 and NS5 components of the polyprotein and relatively poor responses were elicited against NS4 and the core components. IFN-gamma responses, however, were induced against all of the individual components of the polyprotein. These data suggest that the HCV polyprotein delivered with adjuvants induces broad B- and T-cell responses and could be a vaccine candidate against HCV.

Two-Year Reduction of Panel Reactive Human Leukocyte Antigen Antibodies in Children Receiving Mycophenolate Mofetil After Valved Allograft Placement

Allografts used in the repair of congenital heart defects in children induce a persistent broad HLA antibody response. We have previously shown that a 3-month course of mycophenolic mofetil (MMF) significantly reduces the HLA class I antibody response to valved allograft implantation in children. The purpose of this study was to determine if this reduction in HLA antibody persists after discontinuation of MMF. We conducted follow-up (mean 2 +/- 0.5 years) of seven patients who had received allograft placement for repair of congenital heart defects. These patients received 3 months of immunosuppression with MMF following allograft implantation. When compared to historical controls, patients who received MMF following surgery showed a significantly decreased HLA class I antibody response at 2 years postimplantation. This study demonstrates the ability to persistently alter the HLA class I antibody response using 3 months of MMF following allograft implantation in children.

Multiclade Human Immunodeficiency Virus Type 1 Envelope Immunogens Elicit Broad Cellular and Humoral Immunity in Rhesus Monkeys

The development of a human immunodeficiency virus type 1 (HIV-1) vaccine that elicits potent cellular and humoral immune responses recognizing divergent strains of HIV-1 will be critical for combating the global AIDS epidemic. The present studies were initiated to examine the magnitude and breadth of envelope (Env)-specific T-lymphocyte and antibody responses generated by vaccines containing either a single or multiple genetically distant HIV-1 Env immunogens. Rhesus monkeys were immunized with DNA prime-recombinant adenovirus boost vaccines encoding a Gag-Pol-Nef polyprotein in combination with either a single Env or a mixture of clade-A, clade-B, and clade-C Envs. Monkeys receiving the multiclade Env immunization developed robust immune responses to all vaccine antigens and, importantly, a greater breadth of Env recognition than monkeys immunized with vaccines including a single Env immunogen. All groups of vaccinated monkeys demonstrated equivalent immune protection following challenge with the pathogenic simian-human immunodeficiency virus 89.6P. These data suggest that a multicomponent vaccine encoding Env proteins from multiple clades of HIV-1 can generate broad Env-specific T-lymphocyte and antibody responses without antigenic interference. This study demonstrates that it is possible to generate protective immune responses by vaccination with genetically diverse isolates of HIV-1.

Stratification of type 1 diabetes risk on the basis of islet autoantibody characteristics.

Family history of type 1 diabetes and autoantibodies to the islet antigens insulin (IAA), glutamate decarboxylase (GADA), and the protein tyrosine phosphatase-like protein IA-2 (IA-2A) are strong predictors of type 1 diabetes, but the rate of progression to diabetes in multiple islet autoantibody-positive relatives varies widely. We asked whether detailed characterization of islet autoantibodies that included determination of titer, epitope specificity, and IgG subclass would improve diabetes prediction in a large cohort of autoantibody-positive relatives. The study shows a strong association between risk and high titer, broad antibody responses to IA-2 and insulin. The highest risks were associated with high-titer IA-2A and IAA, IgG2, IgG3, and/or IgG4 subclass of IA-2A and IAA, and antibodies to the IA-2-related molecule IA-2beta. Using models based on these antibody characteristics, autoantibody-positive relatives can be classified into groups with risks of diabetes ranging from 7 to 89% within 5 years.

Tat HIV-1 Primary and Tertiary Structures Critical to Immune Response Against Non-homologous Variants

Clinical studies show that in the absence of anti-retroviral therapy an immune response against the human immunodeficiency virus type 1 (HIV-1), transacting transcriptional activator (Tat) protein correlates with long term non-progression. The purpose of this study is to try to understand what can trigger an effective immune response against Tat. We used five Tat variants from HIV strains identified in different parts of the world and showed that mutations of as much as 38% exist without any change in activity. Rabbit sera were raised against Tat variants identified in rapid-progressor patients (Tat HXB2, a European variant and Tat Eli, an African variant) and a long term non-progressor patient (Tat Oyi, an inactive African variant). Enzyme-linked immunosorbent assay (ELISA) results showed that anti-Tat Oyi serum had the highest antibody titer and was the only one to have a broad antibody response against heterologous Tat variants. Surprisingly, Tat HXB2 was better recognized by anti-Tat Oyi serum compared with anti-Tat HXB2 serum. Western blots showed that non-homologous Tat variants were recognized by antibodies directed against conformational epitopes. This study suggests that the primary and tertiary structures of the Tat variant from the long term non-progressor patient are critical to the induction of a broad and effective antibody response against Tat.

HIV vaccine produces broad antibody response

HIV vaccine produces broad antibody response

Glycosylated Ectodomain of the Human Thyrotropin Receptor Induces Antibodies Capable of Reacting with Multiple Blocking Antibody Epitopes

Recently, we showed that the glycosylated ectodomain of the human thyrotropin receptor (hET-gp) reacts with autoantibodies from autoimmune thyroid disease (AITD) patients' sera. To better understand the effects of glycosylation of thyrotropin receptor (TSHR) in antibody induction, we immunized rabbits with hET-gp protein. The rabbits developed relatively high titers of antibodies with highly potent TSH binding inhibitory immunoglobulin (TBII) and thyroid stimulatory blocking antibody (TSBAb) activities. Both the hET-gp and a nonglycosylated ectodomain of the human TSHR (hETSHR) protein significantly reversed the TBII as well as TSBAb activity. Based on the ability of synthetic peptides to significantly reverse the functional activity of these rabbit antisera, we identified three discrete regions of the TSHR, represented by amino acids 202–221, 292–311 and 367–386, as TBII epitopes and four regions represented by amino acids 352–371, 367–386, 382–401 and 392–415 as TSBAb epitopes. These data demonstrate that rabbit antibodies that bind to amino acids 367–386 mediate their TSBAb activity by inhibiting the binding of TSH to TSHR; whereas, antibodies to regions 352–415, excluding aa 367–386, exert their TSBAb activity by affecting a step subsequent to TSH binding. Coincident with the elevation of TBII and TSBAb activity, serum total T4 levels declined and thus suggested that the antibodies exerted functional effects on thyroid in vivo. Together, these data demonstrate that glycosylated hET-gp protein is a more potent immunogen and it can induce a broader antibody response directed against multiple TBII and TSBAb epitopes.

Persistence of Human Leukocyte Antigen (HLA) Antibodies After One Year in Children Receiving Cryopreserved Valved Allografts

This study shows that the broad anti-HLA antibody response against cryopreserved valved allografts used for surgical repair of congenital heart disease persists beyond 1 year after implantation. In 3 patients, there were clearly defined HLA antibody specificities consistent with the HLA phenotypes of the patients, i.e., the panel-reactive antibody was directed against major alloantigen groups that were not expressed by the antibody responders.

Immunoblot detection of oligoclonal anti‐myelin basic protein IgG antibodies in cerebrospinal fluid in multiple sclerosis

Migration properties and occurrence of antibodies against myelin basic protein (MBP) in paired CSF and serum specimens from patients with multiple sclerosis (MS) were demonstrated after agarose isoelectric focusing, immunoblot transfer, and immunoperoxidase staining. Oligoclonal IgG antibody bands directed against MBP were found in the CSF of 9 of 28 patients with MS (32%), but not in the CSF of any of 34 patients with other neurologic diseases. No serum showed anti-MBP antibody bands. The CSF anti-MBP antibodies migrated to the anodal region of the IgG area in a different fashion from oligoclonal IgG and anti-measles IgG antibodies, which were detected in parallel. The anti-MBP bands were transient in three of seven patients whom we studied consecutively. Enzyme-linked immunosorbent assay (ELISA) of serum and CSF for detection of IgG reactivity against MBP showed absorbance values above 2 standard deviations of controls in 44% of the MS patients and in 21% of those with other neurologic diseases. Results of this assay correlated partly with those of the immunoblot assay. ELISA positive and immunoblot negative results might be due to a broad polyclonal anti-MBP antibody response.

Both species of chlamydia and two biovars of Chlamydia trachomatis stimulate mouse B lymphocytes.

We have investigated the ability of both species of chlamydiae (C. trachomatis and C. psittaci), two major biovars of C. trachomatis (lymphogranuloma venereum and trachoma), and the two developmental forms of chlamydia (reticulate and elementary bodies) to stimulate murine spleen lymphocytes. All of these forms of the bacteria induce potent proliferation and differentiation to plaque-forming cells by B lymphocytes in vitro. Chlamydiae induce a broad antibody response, suggesting that stimulation is polyclonal in nature. Although all chlamydiae possess a lipopolysaccharide (LPS) genus-specific molecule similar to LPS found on Re mutant enterobacteria, polyclonal B cell stimulation is likely caused by molecules other than LPS, since i) polymyxin B failed to inhibit chlamydia-induced immunostimulation and ii) C3H/HeJ mice (LPS nonresponders) produced normal numbers of PFC after culture with chlamydia (but not LPS). Thus, a cross-species moiety that is not LPS is responsible for polyclonal stimulation by chlamydia. Because these bacteria can exist in latent forms in an animal, and all forms are immunostimulatory, the question of whether these bacteria can alter immune responses if released during other infections or immunizations has been raised.

THE PREVALENCE AND POSSIBLE SIGNIFICANCE OF IgD GRANULOCYTE‐SPECIFIC ANTINUCLEAR ANTIBODIES IN NEUTROPENIC AND NON‐NEUTROPENIC CASES OF RHEUMATOID ARTHRITIS

Sera from 21 rheumatoid arthritis patients with accompanying neutropenia (less than or equal to 2000 neutrophils/microliter) and 45 rheumatoid arthritis patients without neutropenia were studied for the occurrence of IgD granulocyte-specific antinuclear antibodies. Such antibodies were found in 67 per cent of the neutropenic and 18 per cent of the non-neutropenic cases (p less than 0.001). The titres of IgD granulocyte-specific antinuclear antibodies varied independently of the titres of IgD and complement-fixing granulocyte-specific antinuclear antibodies, but showed some covariation with granulocyte-specific antinuclear antibodies of the IgA and IgM classes most probably reflecting a broad polyclonal antibody response to phagocyte nuclear antigens in the serologically highly active neutropenic cases. Gel filtration studies on selected sera containing IgD granulocyte-specific antinuclear antibodies indicated participation of these antibodies in immune complexes. It is thus possible that IgD granulocyte-specific antinuclear antibodies may have some significance for the rheumatoid inflammatory processes.

Experimental infection of chimpanzees with dengue viruses.

Abstract Single strains of dengue (DEN) virus types 1–4 at low passage levels after isolation in Thailand, New Caledonia or the Philippine Islands produced no overt, febrile or hematologically detectable subclinical disease in chimpanzees inoculated subcutaneously and intradermally. After primary inoculation, viremias occurred usually from days 2 or 3 through days 5–7. Virus was detected in serum with about equal frequencies by either mosquito inoculation or LLCMK2 monkey kidney cell cultural technics, although mosquito inoculations usually yielded higher titers of virus. After secondary challenge with DEN 2 virus 26 mo after primary infection with DEN 1, 2, 3 or 4, viremias lasting 2–4 days occurred during days 3–6 in one or two chimpanzees originally infected with DEN 1 or DEN 4, but not with DEN 2 or DEN 3. Primary homologous DEN hemagglutination-inhibition (HI) antibody responses were greater than heterologous in pairs of young chimpanzees born in the U.S.A. and inoculated with DEN 3 or DEN 4. However, primary homologous DEN HI antibody responses were similar to heterologous responses in five adult chimpanzees from Africa inoculated with DEN 1, 2, 3 or 4 viruses and in three young chimpanzees born in the U.S.A. and inoculated with DEN 2 virus. Homologous and heterologous neutralizing (N) antibodies were found in serum 4–6 wk after primary infection of adult chimpanzees, but only homologous DEN 2 N antibodies were detected in three young chimpanzees. Preexisting antibodies to other flaviviruses were found in sera of some chimpanzees from Africa, and broad flavivirus anamnestic HI antibody responses occurred after primary inoculations of DEN viruses. Broad flavivirus HI antibody responses were also observed after primary DEN inoculations in three young chimpanzees born and housed near New York City and without preexisting flavivirus antibodies. Following secondary challenge with DEN 2 virus, HI and complement fixation antibody responses occurred to all DEN types and to related flaviviruses.

Immunological Studies with Group B Arthropod-Borne Viruses

SummaryHemagglutination-inhibiting antibody responses with selected Group B arthropod-borne virus antigens were studied following administration of living attenuated 17D strain yellow fever vaccine to human subjects who had (a) no previous Group B arbovirus experience, (b) only 17D strain vaccine previously, (c) previous inapparent natural Japanese encephalitis virus infection and (d) complex Group B arbovirus experience previously. The results indicate the following: 1. Previous homologous or heterologous immunological experience did not suppress the yellow fever HI response to the living vaccine. On the contrary, the response was sometimes enhanced. 2. Heterologous HI responses depended upon the nature of the previous Group B arbovirus experience. (a) Subjects with no prevaccination Group B arbovirus experience developed a limited array of low-titered heterologous antibodies. (b) Subjects previously vaccinated with the 17D strain but with no other Group B arbovirus experience exhibited limited intensification and broadening of heterologous HI antibodies. (c) Subjects who had experienced some other Group B virus infection prior to yellow fever vaccination displayed a broad heterologous antibody response producing at times significantly higher HI antibody titers to other viruses. 3. Subjects with relatively low, but not with high, yellow fever neutralization indices prior to revaccination tended to show a significant increase in neutralizing antibody level in response to the vaccines.