Antibody Responses In Monkeys Research Articles

Stabilization of HIV-1 Envelope in the CD4-bound Conformation through Specific Cross-linking of a CD4 Mimetic

CD4 binding on gp120 leads to the exposure of highly conserved regions recognized by the HIV co-receptor CCR5 and by CD4-induced (CD4i) antibodies. A covalent gp120-CD4 complex was shown to elicit CD4i antibody responses in monkeys, which was correlated with control of the HIV virus infection (DeVico, A., Fouts, T., Lewis, G. K., Gallo, R. C., Godfrey, K., Charurat, M., Harris, I., Galmin, L., and Pal, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 17477-17482). Because the inclusion of CD4 in a vaccine formulation should be avoided, due to potential autoimmune reactions, we engineered small sized CD4 mimetics (miniCD4s) that are poorly immunogenic and do not induce anti-CD4 antibodies. We made covalent complexes between such an engineered miniCD4 and gp120 or gp140, through a site-directed coupling reaction. These complexes were recognized by CD4i antibodies as well as by the HIV co-receptor CCR5. In addition, they elicit CD4i antibody responses in rabbits and therefore represent potential vaccine candidates that mimic an important HIV fusion intermediate, without autoimmune hazard.

145 The V1/V2 Loop Region of Simian-Human Immunodeficiency Virus Envelope gp120 is the Major Determinant of the Strain Specificity of the Neutralizing Antibody Response

Plasma samples from individuals infected with human immunodeficiency virus type 1 (HIV-1) are known to be highly strain-specific in their ability to neutralize HIV-1 infectivity. Such plasma samples exhibit significant neutralizing activity against autologous HIV-1 isolates but typically exhibit little or no activity against heterologous strains. Monkeys infected with the simian-human immunodeficiency virus (SHIV) clone DH12 generated antibodies that neutralized SHIV DH12 but not SHIV KB9. Conversely, antibodies from monkeys infected with the SHIV clone KB9 neutralized SHIV KB9 but not SHIV DH12. To investigate the role of the variable loops of the HIV-1 envelope glycoprotein gp120 in determining this strain specificity, variable loops 1 and 2 (V1/V2), 3 (V3) or 4 (V4) were exchanged individually or in combination between SHIV DH12 and SHIV KB9. Despite the fact that both parental viruses exhibited significant infectivity and good replication in the cell lines examined, three of the ten variable loop chimeras exhibited such poor infectivity that they could not be used further for neutralization assays. These results indicate that a variable loop that is functional in the context of one particular envelope background will not necessarily function within another. The remaining seven replication-competent chimeras allowed for unambiguous assignment of the sequences principally responsible for the strain specificity of the neutralizing activity present in SHIV-positive plasma. Exchange of the V1/V2 loop sequences conferred a dominant loss of sensitivity to neutralization by autologous plasma and gain of sensitivity to neutralization by heterologous plasma. Substitution of V3 or V4 had little or no effect on the sensitivity to neutralization. These data demonstrate that the V1/V2 region of HIV-1 gp120 is principally responsible for the strain specificity of the neutralizing antibody response in monkeys infected with these prototypic SHIVs.

Importance of the V1/V2 Loop Region of Simian-Human Immunodeficiency Virus Envelope Glycoprotein gp120 in Determining the Strain Specificity of the Neutralizing Antibody Response

Plasma samples from individuals infected with human immunodeficiency virus type 1 (HIV-1) are known to be highly strain specific in their ability to neutralize HIV-1 infectivity. Such plasma samples exhibit significant neutralizing activity against autologous HIV-1 isolates but typically exhibit little or no activity against heterologous strains, although some cross-neutralizing activity can develop late in infection. Monkeys infected with the simian-human immunodeficiency virus (SHIV) clone DH12 generated antibodies that neutralized SHIV DH12, but not SHIV KB9. Conversely, antibodies from monkeys infected with the SHIV clone KB9 neutralized SHIV KB9, but not SHIV DH12. To investigate the role of the variable loops of the HIV-1 envelope glycoprotein gp120 in determining this strain specificity, variable loops 1 and 2 (V1/V2), V3, or V4 were exchanged individually or in combination between SHIV DH12 and SHIV KB9. Despite the fact that both parental viruses exhibited significant infectivity and good replication in the cell lines examined, 3 of the 10 variable-loop chimeras exhibited such poor infectivity that they could not be used further for neutralization assays. These results indicate that a variable loop that is functional in the context of one particular envelope background will not necessarily function within another. The remaining seven replication-competent chimeras allowed unambiguous assignment of the sequences principally responsible for the strain specificity of the neutralizing activity present in SHIV-positive plasma. Exchange of the V1/V2 loop sequences conferred a dominant loss of sensitivity to neutralization by autologous plasma and a gain of sensitivity to neutralization by heterologous plasma. Substitution of V3 or V4 had little or no effect on the sensitivity to neutralization. These data demonstrate that the V1/V2 region of HIV-1 gp120 is principally responsible for the strain specificity of the neutralizing antibody response in monkeys infected with these prototypic SHIVs.

Maturation of envelope-specific antibody responses to linear determinants in monkeys inoculated with attenuated SIV.

We have previously used a panel of quantitative and qualitative serological assays to define a lengthy and complex maturation of envelope-specific antibody responses in monkeys experimentally infected with attenuated simian immunodeficiency virus (SIV) that is closely associated with the temporal development of enduring and protective immunity to experimental virus challenge. To elucidate in more detail the changes in antibody specificity associated with this maturation, we describe here 'domain-specific' serological studies to characterize the evolution of antibody responses to defined linear determinants of the SIV envelope protein. The results of these studies reveal for the first time distinguishing differences in the evolution of antibody populations to distinct envelope peptide segments, as determined by measurements of antibody titer and avidity, indicating different patterns of antibody maturation to distinct linear envelope antigenic determinants. Thus, these data demonstrate the potential for domain-specific serology to produce a high-resolution characterization of SIV-specific antibody responses that can be used to evaluate experimental vaccine responses and to identify potential immune correlates of protection.

Human immune responses to the highly repetitive Plasmodium falciparum antigen Pf332.

The B and T cell responses to EB200, a repetitive part of the Plasmodium falciparum antigen Pf332, were examined in malaria-exposed Senegalese adults. Most donors had high levels of antibodies to recombinant EB200 and 17 overlapping peptides spanning EB200. Taking proliferation and/or cytokine (interferon-gamma and interleukin-4) production as a measure of T cell activation, eight of the EB200-derived peptides induced responses in > 40% of the donors tested. There was no general association between the different types of T cell responses measured, emphasizing the importance of including multiple parameters when analyzing T cell responses and suggesting that EB200 induces functionally distinct T cell responses. The most efficient peptide for induction of proliferative responses was one previously shown to induce T cell responses in five different H-2 congenic mouse strains primed with EB200, suggesting that this is a universal T cell epitope. The presence of multiple B and T cell epitopes in EB200, widely recognized by humans, is important since EB200 has been shown to elicit protective antibody responses in monkeys and may be considered for inclusion in malaria subunit vaccines.

Evolution of envelope-specific antibody responses in monkeys experimentally infected or immunized with simian immunodeficiency virus and its association with the development of protective immunity.

Previous studies of attenuated simian immunodeficiency virus (SIV) vaccines in rhesus macaques have demonstrated the development of broad protection against experimental challenge, indicating the potential for the production of highly effective immune responses to SIV antigens. However, the development of this protective immune status was found to be critically dependent on the length of time postvaccination with the attenuated virus strain, suggesting a necessary maturation of immune responses. In this study, the evolution of SIV envelope-specific antibodies in monkeys experimentally infected with various attenuated strains of SIV was characterized by using a comprehensive panel of serological assays to assess the progression of antibodies in longitudinal serum samples that indicate the development of protective immunity. In parallel studies, we also used the same panel of antibody assays to characterize the properties of SIV envelope-specific antibodies elicited by inactivated whole-virus and envelope subunit vaccines previously reported to be ineffective in producing protective immunity. The results of these studies demonstrate that the evolution of protective immunity in monkeys inoculated with attenuated strains of SIV is associated with a complex and lengthy maturation of antibody responses over the first 6 to 8 months postinoculation, as reflected in progressive changes in antibody conformational dependence and avidity properties. The establishment of long-term protective immunity at this time in general parallels the absence of further detectable changes in antibody responses and a maintenance of relatively constant antibody titer, avidity, conformational dependence, and the presence of neutralizing antibody for at least 2 years postinoculation. In contrast to the mature antibody responses elicited by the attenuated SIV vaccines, the whole-virus and envelope subunit vaccines in general elicited only immature antibody responses characterized by poor reactivity with native envelope proteins, low avidity, low conformational dependence, and the absence of neutralization activity against the challenge strain. Thus, these studies establish for the first time an association between the effectiveness of experimental vaccines and the capacity of the vaccine to produce a mature antibody response to SIV envelope proteins and further indicate that a combination of several antibody parameters (including titer, avidity, conformational dependence, and virus neutralization) are superior to any single antibody parameter as prognostic indicators to evaluate candidate AIDS vaccines.

Salivary, nasal, genital, and systemic antibody responses in monkeys immunized intranasally with a bacterial protein antigen and the Cholera toxin B subunit

Previous attempts to induce mucosal antibodies in rhesus monkeys by enteric immunization have resulted in only modest and short-lived responses, dominated by immunoglobulin M (IgM) antibodies in the plasma. In this study, two groups of rhesus monkeys were immunized intranasally three times at 2-week intervals with a bacterial protein antigen (AgI/II) either chemically coupled to or mixed with the B subunit of cholera toxin (CT), a known potent mucosal immunogen and carrier for other immunogens. Cells secreting antibodies, predominantly of the IgA isotype, to AgI/II and to CT were detected in the peripheral blood 1 week after each immunization, indicating the dissemination of IgA-secreting precursor cells through the mucosal immune system. IgG and, to a lesser extent, IgA antibodies to both proteins were induced in the plasma commencing after the second immunization. Plasma IgE concentrations and IgE antibody levels were not consistently raised during the immunization period. IgA antibodies were found in nasal and vaginal washes. Nasal IgG but not IgA antibodies showed a significant positive correlation with plasma IgG antibody levels, suggesting that they were largely derived by transudation from the circulation. Analysis of the molecular form of vaginal IgA indicated that both secretory and monomeric forms of IgA were present in various proportions. Furthermore, neither IgG nor IgA antibodies in vaginal washes were correlated with plasma antibody responses, suggesting the contribution of locally synthesized antibodies of both isotypes. Comparison of the responses between the two groups of animals showed only sporadic significant differences, indicating that intranasal immunization with AgI/II either coupled to or mixed with the B subunit of CT was equally effective at inducing generalized IgA antibody responses in the mucosal immune system and predominantly IgG antibodies in the plasma.

Detection of antibodies to the major outer membrane protein of Chlamydia trachomatis using an in vitro transcription-translation radioimmunoprecipitation assay

A radioimmunoprecipitation assay (RIPA) using in vitro translated protein was developed to measure serum antibodies to the major outer membrane protein (MOMP) of C. trachomatis. Polyclonal animal antisera to C. trachomatis serovars C, E and F reacted to serogroup-specific determinants by RIPA, as compared to species-specific epitopes by Western blot. Antibody responses in monkeys immunized systemically or mucosally with a candidate MOMP vaccine were assessed by RIPA and ELISA with elementary bodies (EB-ELISA). Unlike EB-ELISA, RIPA showed a significant difference in pre-challenge antibody levels between systemically and mucosally immunized animals. Additionally, only RIPA showed a significant inverse correlation between antibody level at time of challenge and microbiologic response measured as median inclusion forming units (IFU) in culture (r = −0.89; P < 0.001). RIPA using in vitro translated proteins is a useful method to measure antibody responses to specific C. trachomatis antigens and may be more informative than EB-ELISA and Western blot for assessing the immunogenicity of MOMP vaccines.

Comparison of the immunogenicity of recombinant and pituitary human growth hormone in rhesus monkeys

The relative concentrations of antibodies produced in monkeys against three forms of human growth hormone (hGH) were determined using an antigen-specific avidin/biotin ELISA assay. Monkeys were treated in two separate 90-day studies with recombinant methionyl-hGH (met-hGH) and pituitary-derived hGH (pit-hGH) (Study 1) and recombinant natural sequence hGH (Study 2). The lowest dose was equal to the expected therapeutic dose of 0.1 IU/kg. Sixty-nine percent of monkeys treated with pit-hGH and 81% of those treated with met-hGH developed detectable anti-hGH responses. The magnitudes of the responses exhibited wide animal to animal variability, were not markedly related to dose or sex, and were lower than levels obtained in monkeys immunized with hGH in Freund's adjuvant. In contrast, the incidence of antibody responses in monkeys treated with natural sequence hGH was lower (23% in one experiment and 5% in a replicate experiment) and took longer to develop. Antibody concentrations were lower, on average, than in those animals treated with met- or pit-hGH. These results are in accord with those observed clinically, thus supporting the use of the monkey model to predict the relative immunogenicity of some proteins in humans.

Comparison of the Immunogenicity of Recombinant and Pituitary Human Growth Hormone in Rhesus Monkeys

The relative concentrations of antibodies produced in monkeys against three forms of human growth hormone (hGH) were determined using an antigen-specific avidin/biotin ELISA assay. Monkeys were treated in two separate 90-day studies with recombinant methionyl-hGH (met-hGH) and pituitary-derived hGH (pit-hGH) (Study 1) and recombinant natural sequence hGH (Study 2). The lowest dose was equal to the expected therapeutic dose of 0.1 IU/kg. Sixty-nine percent of monkeys treated with pit-hGH and 81% of those treated with met-hGH developed detectable anti-hGH responses. The magnitudes of the responses exhibited wide animal to animal variability, were not markedly related to dose or sex, and were lower than levels obtained in monkeys immunized with hGH in Freund's adjuvant. In contrast, the incidence of antibody responses in monkeys treated with natural sequence hGH was lower (23% in one experiment and 5% in a replicate experiment) and took longer to develop. Antibody concentrations were lower, on average, than in those animals treated with met- or pit-hGH. These results are in accord with those observed clinically, thus supporting the use of the monkey model to predict the relative immunogenicity of some proteins in humans.

Radioimmunoassay of class-specific antibodies to Streptococcus mutans in monkey serum and saliva

A radioimmunoassay (RIA) has been developed to measure class-specific antibodies to Streptococcus mutans in the serum and saliva of monkeys ( Macaca fascicularis). Anti-human immunoglobulin antibodies purified by affinity chromatography on immobilised monkey immunoglobulins and labelled with 125I were employed. Formolised cells of S. mutans and an extract of culture supernatant adsorbed to polystyrene wells were used as solid-phase antigens. The coefficients of variation for IgG, IgA and IgM assays were ⩽ 10% for both antigen systems. Two monkeys were immunised with formolised cells of S. mutans by subcutaneous injection and subsequent instillation of bacterial cells into their right parotid ducts. IgG, IgA and IgM antibody responses to S. mutans in samples of serum and saliva were quantitated by RIA. Immobilisation of purified components of S. mutans on polystyrene wells enabled the measurement of antibody response to a number of antigens to be made. The RIA is a sensitive, reproducible and quantitative method of measuring serum and salivary antibody responses in monkeys.

Immunological properties of dinitrophenyl derivatives of beta-subunit of the human chorionic gonadotropin

Four different derivatives with varying proportions of dinitrophenyl (DNP) groups tagged covalently to β-subunit of HCG (DNP (1)-β-HCG, DNP (3.3)-β-HCG, DNP (6)-β-HCG and DNP (10)-β-HCG) have been prepared. Their immunogenicity as well as the reactivity of antibodies formed in mice, rabbit and rhesus monkey have been studied. The derivatives give rise to the formation of antibodies in all species tested which react with HCG and in some species with DNP moieties. The anti-DNP response increases with derivatives bearing more than 3–4 DNP groups per molecule of β-HCG. On the other hand the anti-HCG response diminishes with derivatives bearing higher than 3–4 DNP groups per β-HCG molecule. The antibody response in monkeys is of long duration (> 14 months). The association constants for binding with HCG are of the order of 4 × 10 9 to 6 × 10 10 L/mole. the affinity of the antibodies tends to increase with time.

Effects of antigen-antibody complexes on the primary immune response in Rhesus monkeys.

A model for the enhancement of the primary humoral immune response of rhesus monkeys to marginal or weakly antigenic vaccines is presented. Our procedure used the complexing of formalin-inactivated Venezuelan equine encephalomyelitis (VEE) virus vaccine with specific, homologous, immune gamma globulin (IgG) at equivalence. Equivalence was determined by combining the concentrated, isotopically labeled ((3)H) virus with various concentrations of specific Sephadex-fractionated IgG. Enhancement of VEE virus antibody response in monkeys was obtained from preparations containing a marginal concentration of antigen that was complexed at equivalence with homologous IgG as compared with antigen alone. Protection in Swiss mice closely paralleled the antibody response pattern observed in monkeys, since complexes at equivalence provided 30 to 50% greater protection against challenge than antigen alone.

Yellow fever vaccine. V. Antibody response in maonkeys inoculated with graded doses of the 17D vaccine.

A dosage equal to or greater than approximately 3.4 Dex (decimal exponent, log(10)) weanling mouse intracerebral 50% lethal dose (LD(50)) was sufficient to elicit a yellow fever antibody response, as determined by the plaque neutralization (PN) test, in better than 90% of vaccinated rhesus monkeys. Lower dosages were progressively less effective in terms of PN titers and the PN and hemagglutination-inhibition serological conversion rates observed. A dose of between 3.4 and 4.2 Dex weanling mouse intracerebral LD(50), or one-tenth to one times the dosage recommended for man, provided an optimal antibody response in monkeys. In rhesus monkeys, in contrast to the findings for man, pre-existing yellow fever antibody did not interfere with the antibody response to yellow fever vaccine. The PN test was felt to be a more sensitive and specific indicator of yellow fever antibody in rhesus monkeys after vaccination than the hemagglutination inhibition or complement fixation tests.

Yellow Fever Vaccine. V. Antibody Response in Monkeys Inoculated with Graded Doses of the 17D Vaccine

A dosage equal to or greater than approximately 3.4 Dex (decimal exponent, log(10)) weanling mouse intracerebral 50% lethal dose (LD(50)) was sufficient to elicit a yellow fever antibody response, as determined by the plaque neutralization (PN) test, in better than 90% of vaccinated rhesus monkeys. Lower dosages were progressively less effective in terms of PN titers and the PN and hemagglutination-inhibition serological conversion rates observed. A dose of between 3.4 and 4.2 Dex weanling mouse intracerebral LD(50), or one-tenth to one times the dosage recommended for man, provided an optimal antibody response in monkeys. In rhesus monkeys, in contrast to the findings for man, pre-existing yellow fever antibody did not interfere with the antibody response to yellow fever vaccine. The PN test was felt to be a more sensitive and specific indicator of yellow fever antibody in rhesus monkeys after vaccination than the hemagglutination inhibition or complement fixation tests.

Yellow fever vaccine. II. Antigenicity and neurovirulence of a vaccine seed free from avian leukosis virus.

Avian leukosis virus (ALV)-free candidate primary and secondary seed lots were indistinguishable from corresponding ALV-contaminated lots with respect to (i) potency as measured by titration in newborn and weanling mice and in the MA-104 plaque system, (ii) degree of viscerotropism as measured by viremia in monkeys, (iii) neurotropism as determined by the monkey neurovirulence test, and (iv) potency as determined by antibody response in monkeys inoculated by the intracerebral route.

Yellow Fever Vaccine. II. Antigenicity and Neurovirulence of a Vaccine Seed Free from Avian Leukosis Virus

Avian leukosis virus (ALV)-free candidate primary and secondary seed lots were indistinguishable from corresponding ALV-contaminated lots with respect to (i) potency as measured by titration in newborn and weanling mice and in the MA-104 plaque system, (ii) degree of viscerotropism as measured by viremia in monkeys, (iii) neurotropism as determined by the monkey neurovirulence test, and (iv) potency as determined by antibody response in monkeys inoculated by the intracerebral route.

Trachoma-studies of etiology, laboratory diagnosis, and prevention.

Material from 116 human eyes infected with trachoma was used successfully for the isolation of viruses by culturing in the yolk sac of chick embryos. Five strains of virus were isolated. All five killed embryos on second passage, and all five fixed complement with both serum from patients with lymphogranuloma venereum and a guinea pig psittacosis antiserum. The virus thus made available for laboratory study was used in tests for sensitivity to drugs. Penicillin, sulfisoxazole, and oxytetracycline were effective against the virus even in the smallest amounts used. The virus produced typical acute follicular conjunctivitis in six human volunteers. A vaccine was produced from the virus and elicited a significant antibody response in monkeys and human subjects. Evidence that the vaccine had both preventive and curative value was obtained.