Initial Antibody Response Research Articles

Immune Responses in Cattle Inoculated with Mycobacterium bovis , Mycobacterium tuberculosis , or Mycobacterium kansasii

Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare the antigen-specific immune responses to various patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis infection) and colonization without pathology (M. tuberculosis infection) to no colonization or pathology (M. kansasii infection). Delayed-type hypersensitivity and gamma interferon responses were elicited by each mycobacterial inoculation; however, the responses by the M. bovis- and M. tuberculosis-inoculated animals exceeded those of the M. kansasii-inoculated animals. Specific antibody responses were detected in all M. tuberculosis- and M. bovis-inoculated cattle 3 weeks after inoculation. From 6 to 16 weeks after M. tuberculosis inoculation, the antibody responses waned, whereas the responses persisted with M. bovis infection. With M. kansasii inoculation, initial early antibody responses waned by 10 weeks after inoculation and then increased 2 weeks after the injection of purified protein derivative for the skin test at 18 weeks after challenge. These findings indicate that antibody responses are associated with the antigen burden rather than the pathology, cellular immune responses to tuberculin correlate with infection but not necessarily with the pathology or bacterial burden, and exposure to mycobacterial antigens may elicit an antibody response in a presensitized animal.

HIV-1-specific antibody responses during acute and chronic HIV-1 infection

The humoral immune response to HIV-1 throughout infection is comprised of complex mixtures of antibody isotypes with numerous HIV-1 specificities. However, unlike antibody responses to most infections, protective antibody responses are delayed and do not arise until long after HIV-1 latency is established. We review recent data on HIV-1-specific antibody isotypes induced following HIV-1 transmission: to understand the effects of HIV-1 on B cell and T cell effector responses, to understand the timing of the rise and fall of different anti-HIV-1 antibodies and to understand how antibodies could contribute to protective immunity if they were either pre-existing or elicited immediately after HIV-1 transmission. Studies of the earliest events following infection by the transmitted/founder virus have recently revealed that early destruction of B cell generative microenvironments may be responsible for delay of potentially protective anti-HIV-1 antibody responses. Unlike the initial CD8 T cell response to HIV-1, the initial induced antibody response is usually ineffective in controlling virus replication during acute HIV-1 infection. The antibody isotypes and specificities elicited during HIV-1 infection can provide a window into deciphering the detrimental effects of HIV-1 on B cell and T cell responses. Additionally, further characterization of the virus inhibitory capabilities of anti-HIV-1 antibody isotypes can define the spectrum of potential protective HIV-1 antibodies that could be readily elicited by experimental vaccines and adjuvants.

FcγRIII Mediates Immunoglobulin G-Induced Interleukin-10 and Is Required for ChronicLeishmania mexicanaLesions

FcRgamma and interleukin-10 (IL-10) are both required for chronic disease in C57BL/6 mice with Leishmania mexicana parasite infection. FcRgamma is a component of several different FcRs and may be a component of some T-cell receptors. The initial antibody response to L. mexicana is an immunoglobulin G1 (IgG1) response, and IgG1 preferentially binds to FcgammaRIII in other systems. To begin to dissect the mechanisms by which FcgammaRs contribute to chronic disease, we infected FcgammaRIII knockout (KO) mice with L. mexicana. We show that FcgammaRIII KO mice are resistant to L. mexicana infection, resolving lesions in association with a stronger gamma interferon response, similar to IL-10 KO mice, with parasite control by 12 weeks. We found that the Leishmania-specific IgG response is unaltered in FcgammaRIII KO mice compared with that in wild-type controls. The frequencies of IL-10 production from lymph node CD25(+) CD4(+) T cells are the same in KO and wild-type mice, and depletion of CD25(+) cells did not alter the course of infection, implying that T(reg) cells may not be the mechanism for susceptibility to L. mexicana infection, unlike for L. major infection. However, IL-10 mRNA was greatly diminished in the lesions of FcgammaRIII KO mice compared to that of B6 controls. Furthermore, macrophages from FcgammaRIII KO and FcRgamma KO mice have the same profound defect in IL-10 production induced by IgG-opsonized amastigotes. We also found IL-10-dependent (major) and -independent (minor) inhibition of IL-12 mediated by FcgammaRIII, as well as parasite-mediated inhibition of IL-12 and induction of IL-10, independent of FcgammaR. Our data demonstrate a specific role for FcgammaRIII in suppressing protective immunity in L. mexicana infection, likely through macrophage IL-10 production in the lesion.

PROTECTION AGAINST SCRUB TYPHUS BY A PLASMID VACCINE ENCODING THE 56-KD OUTER MEMBRANE PROTEIN ANTIGEN GENE

The 56-kD outer membrane protein of Orientia tsutsugamushi has previously been shown to be the immunodominant antigen in scrub typhus infections. Its gene was cloned into the DNA vaccine vector pVR1012 as a vaccine candidate (pKarp56). The in vitro expression of this 56-kD antigen by pKarp56 was confirmed in tissue culture by an indirect fluorescence assay and Western blot analysis. The initial antibody responses of mice immunized with varied doses of the pKarp56 were barely detected, but increases were observed after each of three subsequent booster immunizations. Although no protection was observed with a single immunization of pKarp56, after four immunizations, 60% of the mice survived a 1,000 x 50% lethal dose (LD(50)) challenge. These results specifically confirm the importance of the 56-kD protein antigen in protective immunity against O. tsutsugamushi and demonstrate the feasibility of DNA vaccines for the prevention of scrub typhus.

Hepatitis B Vaccination Provides Protection for At Least 15 Years

Hepatitis B Vaccination Provides Protection for At Least 15 Years

27 kDa Fasciola gigantica Glycoprotein for the Diagnosis of Prepatent Fasciolosis in Cattle

An indirect enzyme-linked immunosorbent assay (ELISA) using 27 kDa glycoprotein of Fasciola gigantica has been evaluated for its potential use in the diagnosis of bovine fasciolosis. Following experimental infection of rabbits, F. gigantica infection-induced antibodies were isolated and later used as ligands in affinity chromatography for isolation of infection-induced antibody-specific proteins. Among the five infection-specific proteins isolated, a glycoprotein of 27 kDa was later isolated by second-step purification using concanavalin A matrix. In crossbred cattle receiving different doses of infection (100, 200 and 400 metacercariae), the anti-27 kDa antibodies were detected as early as the 2nd week post infection. No direct correlation between initial dose, antibody response and fluke establishment was recorded. No cross-reaction was noted with the sera of goats experimentally infected with Paramphistomum epiclitum. ELISA with the 27 kDa glycoprotein could be a feasible diagnostic tool for the early detection of bovine fasciolosis.

Antigen levels and antibody titers after DNA vaccination

DNA vaccination generates strong cellular and humoral immunity in animal models. The mechanisms by which plasmid DNA uptake and expression after intramuscular injection lead to immune responses are not well understood. In particular, the importance of antigen expression levels on subsequent antibody immune responses has not been established. We found that a chemiluminescent assay for alkaline phosphatase allows measurement of antigen levels of secreted alkaline phosphatase (SEAP) in vivo after intramuscular injection of a wide range of plasmid doses. The mice produced antibodies to the alkaline phosphatase reporter gene and both antigen levels and antibody titers were measured over time. We found that the correlation between initial antigen level and antibody response was high (r = 0.74, p < 0.001) and remained high even after accounting for the dose of plasmid injected (r = 0.61, p < 0.001). The correlation between DNA dose and antibody titer was statistically significant (r = 0.53, p < 0.001) but was reduced to almost zero after we accounted for initial antigen levels. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:474–484, 2001

Consequences for immune function of metabolic adaptations to load

AbstractProlonged periods of stress have been associated with impaired immune function; the experiment reported here investigates a potential link between level of metabolic load and immune function in lactating dairy cattle. A group of 111 Holstein-Friesian dairy cows was used. The cows belonged to one of two genetic lines: a selection line (S) with high genetic merit for fat plus protein yield and an unselected control line (C). The cows were offered one of two silage-based total mixed diets containing either 200 g (LC) or 450 g (HC) of concentrate per kg dry matter. Combination of genetic selection and food gave four groups: S-LC, S-HC, C-LC and C-HC. All cows were inoculated with a live attenuated BHV-1 vaccine soon after parturition and the primary antibody response in whey monitored. The number of BHV-1 antibody positive cows was not significantly different between the four groups; but, the initial antibody response was lower in cows of high genetic merit which were given a low concentrate diet. Statistical analysis demonstrated that the contribution of diet to this effect was highly significant. One year later, again after parturition, the experiment was repeated, this time using serum as the test sample. The average antibody response of the BHV-1 antibody positive cows was not significantly different between the four groups but the number of antibody positive cows was group-dependent. In conclusion, diet type but not genetic merit for high fat plus protein yield made a highly significant contribution to the antibody response of dairy cows to BHV-1 vaccination, both initially and a year later.

Persistence of antibody responses to Haemophilus influenzae type b polysaccharide conjugate vaccine in children with vertically acquired human immunodeficiency virus infection.

Recurrent bacterial sepsis is common in pediatric HIV infection and immunization against Haemophilus influenzae type b (Hib) is recommended. Long term persistence of anti-Hib antibody and the need for, or timing of, a booster dose has not been adequately studied. Immunogenicity during a 12-month period following immunization with Hib-tetanus conjugate vaccine (ACT-HIB; Merieux) was evaluated in 48 vertically HIV-infected children and 36 uninfected children, born to HIV-positive mothers. A titer of anti-Hib polysaccharide antibody of > or = 0.15 microgram/ml was considered to indicate short term and > or = 1 microgram/ml long term protection. At 1 month postvaccination 36 (100%) uninfected and 42 (88%) HIV-infected children achieved titers of > or = 1 microgram/ml. However, by 1 year titers had dropped below this value in 18 (43%) infected compared with only 4 (11%) uninfected children (chi square, 9.7; P = 0.002). Although the rate of fall of antibody titer was greater in uninfected than in infected children, this was no longer the case after adjustment for the 1-month postimmunization titer. The rate of antibody titer decline was not significantly related to HIV disease status or to either the age-related CD4 count at the time of immunization or the change in age-adjusted CD4 count during the 12 months after immunization. Not only was the initial antibody response to Hib conjugate vaccine decreased in children with HIV infection and AIDS but also 1 year later only 57% of the initial responders had persisting titers above the level associated with long term protection. The need for reimmunization of children with HIV infection against Hib requires further evaluation.

Long-term antibody production is sustained by antibody-secreting cells in the bone marrow following acute viral infection.

Acute viral infection of humans induces virus-specific serum antibody production that often persists for decades. To better understand the nature of this long-term antiviral antibody response, we studied antiviral antibody production of mice acutely infected with lymphocytic choriomeningitis virus (LCMV). Although this viral infection is resolved within 2 weeks, virus-specific serum antibody levels were maintained for > 300 days postinfection. The anatomic site of long-term antibody production was identified using an ELISPOT assay to quantitate LCMV-specific antibody-secreting cells (ASC). The initial antiviral ASC response in the spleen peaked at 8 days postinfection and then declined sharply, with less than 10% of the day 8 ASC population remaining after 60 days. Although no LCMV specific ASC were detected in the bone marrow at day 8, virus-specific ASC began accumulating in the bone marrow by 15 days postinfection. By day 60, approximately 10-fold more antiviral ASC were present in the bone marrow than in the spleen, and the bone marrow remained the major site of antibody production for > 10 months postinfection. To further characterize the LCMV-specific antibody response, the relative percentage of each IgG subclass (IgG1, IgG2a, IgG2b, and IgG3) was determined. IgG2a was the predominant IgG subclass produced during LCMV infection, and the IgG subclass profile of virus-specific ASC in the spleen and bone marrow matched the IgG subclass profile of virus-specific IgG in the serum. Following a secondary infection with LCMV, splenic ASC numbers increased rapidly with a peak at 5 days after secondary infection. This was followed by a sharp decline in ASC numbers by day 15. In contrast, virus-specific ASC numbers in bone marrow remained essentially unchanged during the acute phase of the secondary infection but increased approximately twofold at day 15, corresponding to a twofold increase in virus-specific serum antibody levels. These results indicate that following a primary viral infection or upon reexposure to a virus, the initial antibody response occurs in the spleen, but long-term antiviral antibody production is maintained in the bone marrow.

Antigen recognition during progression from acute to chronic infection with a cagA-positive strain of Helicobacter pylori.

We have previously published two reports on acute infection with Helicobacter pylori, one of an adult male and one of a family of four. In the present study, we have isolated H.pylori from each of twin boys in the family and compared these by use of random amplified polymorphic DNA PCR. In addition, we have monitored the antibody response over time of the family and the adult male by Western blotting (immunoblotting) with two different strains of H. pylori as the antigen and by use of a commercial kit. The acutely infected twin boys were infected by an identical strain of H. pylori. The twin boys responded to antigens of 19, 26.5, and 29 kDa 30 days after the initial diagnosis, with recognition of 43-to 49-, 66-, 69-, and 87-kDa antigens by day 63. One twin responded to the CagA antigen on day 63, whereas the other responded on day 857. Antibody to the CagA antigen was not detected by use of the infecting strain, UNSW-RU1. Investigation of UNSW-RU1 revealed the presence of cagA. In two acutely infected adults (one, the father of the boys), the initial response to a 45-kDA antigen was later followed by responses to 19-, 29-, 49-, 60-, 77-, and 84-kDa antigens. Sera from the twins' younger sister, born 17 months after the twins acute episode, indicated that she also had become infected. This report supports intrafamilial transmission of H. pylori. Initial antibody responses in the children were to small-molecular-size antigens; in the adults, the initial responses were to larger-molecular-size antigens. The pattern of the serological response differs according to the antigen used. This has implications in regard to international data comparisons.

Neuroinvasion by simian immunodeficiency virus coincides with increased numbers of perivascular macrophages/microglia and intrathecal immune activation.

During peak viremia and initial antibody response, rhesus macaques infected with pathogenic and nonpathogenic isolates of SIV show distinct differences in viral load and tissue distribution. Animals infected with pathogenic isolates of SIV invariably have virus in the CSF and brain parenchyma by two weeks postinoculation, whereas animals infected with nonpathogenic isolates do not. Mechanisms underlying neuroinvasion by SIV and HIV are unknown, but recruitment of latently infected mononuclear cells from the peripheral circulation (Trojan horse theory) is frequently proposed. Circulating monocytes, from which perivascular macrophage/microglia are derived, are a likely vehicle for cell-associated transport of virus across the blood-brain barrier. This transport and the kinetics of perivascular macrophage/microglial turnover in the CNS likely depend on endothelial and leukocyte adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), which has previously been shown to be upregulated on cerebrovascular endothelium in SIV encephalitis. To investigate the role of peripheral monocyte recruitment into the perivascular macrophage/microglial cell pool at the time of initial viral neuroinvasion, we examined the temporal relationships among perivascular macrophage/microglia density, endothelial VCAM-1 expression and localization of viral nucleic acid in the CNS of macaques acutely infected with pathogenic and nonpathogenic molecular clones of SIV. The concentration of CSF quinolinic acid, a marker of intrathecal immune and macrophage activation, was examined concurrently. We found that significant increases in the density of perivascular macrophages/microglia coincided with viral neuroinvasion and marked elevations in CSF quinolinic acid. Furthermore, combined in situ hybridization and immunohistochemistry demonstrated that infected perivascular cells were macrophages/microglia. These findings provide evidence suggesting that neuroinvasion occurs through an influx of infected monocytes which take up residence in the CNS as perivascular macrophages/microglia. VCAM-1 expression, however, was not clearly correlated with these events, thus its contribution to initial viral neuroinvasion is unclear.

Comparative humoral responses to HIV-1 p24gag and gp120env in subjects from East Africa and the UK

We compared 1616 sera from HIV-1-infected subjects and matched HIV-negative local controls in Uganda, Kenya and the UK. Sera were screened for specific antibody to HIV-1 p24 Gag and gp120 Env proteins and for p24 antigenaemia. In contrast to the UK, the majority of African HIV-1-infected subjects maintained detectable anti-p24 antibodies. However, lower reactivity of anti-p24 was observed in African AIDS patients, compared with those with asymptomatic HIV-1 infection. This reduction in anti-p24 reactivity with more advanced clinical stage was less marked in African HIV-1 infection than in the UK. Correspondingly, p24 antigenaemia was more common in patients with AIDS from the UK than in African patients (65 versus 4%). Reductions in anti-gp120 reactivity were observed in African AIDS patients, compared with the asymptomatic group. However, median reactivity of anti-gp120 in UK patients remained unchanged in both asymptomatic and AIDS subjects. The differences in humoral response to p24 and gp120 between Africa and the UK are semi-quantitative rather than qualitative and could be explained by initial higher antibody response to HIV-1 in African subjects.

Molecular characterization of single memory B cells

Primary antigenic exposure results in an initial antibody response and the T cell-dependent induction of B-cell memory. Memory B-cell differentiation is characterized by somatic hypermutation in antibody variable region genes (V) and selection of B cells expressing high-affinity variants of this antigen receptor. Despite our current understanding of B-cell memory, the origin of memory B cells and the regulation of their differentiation remain elusive. This is largely due to the difficulties in observing and purifying this minor component of the immunized spleen. Further, molecular characterization of memory B cells requires hybridoma formation which restricts analyses to only those clones capable of fusion and does not allow isolation of cells in a normal physiological state. We have therefore developed a unique system which allows isolation and unambiguous enumeration of IgG1+ memory B cells, based on six-parameter flow cytometry, secretion of antibody in clonal cultures and analysis of clonally expressed V genes using the polymerase chain reaction. Here we report that single IgG1+ antigen-binding B cells from an early secondary immune response proliferate in lipopolysaccharide-driven microcultures and produce antigen-specific IgG1 antibodies. Individual B-cell clones in these cultures express somatically mutated heavy chain V genes, confirming their designation as memory B cells. Although isolated memory B cells undergo extensive proliferation in vitro, V gene sequence analysis of their individual progeny shows that further hypermutation does not occur.

Vaccination aginst chronic viral carriage in The Gambia

358 children in the Gambian villages of Keneba and Manduar, where hepatitis B virus (HBV) infection is endemic, were vaccinated with plasma-derived vaccine against HBV according to one of four regimens and followed for up to 4 years. Two regimens by which vaccine was injected intradermally into children between 0 and 4 years old led to peak geometric mean (95% Cl) concentrations of antibody against HBV surface antigen of 270 (202-358) and 555 (418-748) mlU/ml. The third regimen—intramuscular vaccination of children aged between 0 and 4 years—gave geometric mean peak antibody concentrations of 926 (765-1122) mlU/ml. A fourth regimen was intramuscular vaccination of children between 1 and 9 months old, which gave geometric mean antibody concentrations of 5431 (3903-75 456) mlU/ml. Despite these widely divergent responses and a 89% decay in antibody over the first 2 years, vaccination against HBV was 97% effective in preventing chronic infection. Vaccination was less effective in preventing uncomplicated infection: 5·3% of 264 vaccinees in Keneba and 19·1% of 94 vaccinees in Manduar tested positive for antibody to HBV core antigen. These "breakthrough infections" did not differ in frequency between regimens, and were associated with low initial antibody responses and chronic maternal carriage of HBV.

Relation between humoral responses to HIV gag and env proteins at seroconversion and clinical outcome of HIV infection.

To study the contribution of the humoral response to HIV-I at seroconversion to disease outcome after 84 months. A retrospective longitudinal study. Two haemophilia centres in the United Kingdom. 88 Haemophiliac patients infected with HIV-I for whom sera were available from before seroconversion and in whom clinical follow up data were available. Kaplan-Meier survival analysis showed a significant difference between a high titre (greater than 1600) p24 antibody response at seroconversion and prolonged time before the development of HIV related disease (p = 0.0008). In contrast, higher titres of antibody to gp120 at seroconversion (greater than 25,600) correlated with more rapid clinical deterioration (p = 0.025). The first humoral response to HIV proteins at seroconversion is associated with clinical outcome; patients with an initial low titre antibody response to the gagp24 protein have a significantly faster rate of progression to CDC stage IV disease. Patients with a high titre p24 antibody response progress to AIDS more slowly, and these data provide an explanation why p24 antigenaemia is not universally detected in patients with AIDS. It is unclear whether the association between a strong initial p24 antibody response and slower progression of HIV disease is causal and if so whether it is due to viral or host factors.

Kinetics of antibody response to hepatitis B virus determinants and to recombinant vaccines in Italy

The kinetics of antibody response to the group determinant a and subdeterminants d and y of hepatitis B virus were studied after infection and following immunisation with two recombinant DNA yeast-derived hepatitis B vaccines. The initial antibody response was to the subdeterminant epitopes, whereas anti-a antibody, which provides protection against different subtypes of the virus, was not detected for some weeks or months. The delay in the development of anti-a antibody after active immunisation raises important issues of early protection against infection.

Determining Matrilines by Antibody Response to Exotic Antigens

The antibody responses of female Microtus pennsylvanicus inoculated with a series of antigens not normally encountered under field conditions were examined, and the kinetics of maternally acquired antibody loss in their offspring were determined. The initial antibody response in adults was rapid, peaking in 4–9 weeks, and long-lasting, with a half life of 4–5 months. Antibody levels in females were unaffected by parity, and more than one antigen could be given without affecting circulating-antibody titers. Antibody titers could be enhanced with additional inoculations. Maternal antibody in offspring increased until weaning at 3 weeks then declined exponentially. Minimal detectable titers were reached at 7–11 weeks. In many instances, maternal antibody remained detectable even after offspring reached adult (35 g) size. Examination of uninoculated wild-caught voles showed only one of 130 tests produced a “false” positive response. The exotic-antigen technique may be generally applicable for determining maternity in small, secretive mammals.

Antibodies to Plasmodium falciparum sporozoites following a malarial outbreak in a non-endemic area of Sri Lanka

An enzyme-linked immunosorbent assay (ELISA) based on the synthetic peptide (NANP) 40 was used to characterize the sporozoite antibodies in an unusual Plasmodium falciparum outbreak in a non-malarious area in Sri Lanka. A positive antibody response was seen in 62% of patients with their first P. falciparum illness. There was no correlation between sporozoite antibodies and the antibody against blood stages, determined by immunofluorescence assay. The majority (91%) of the patients lost the antibodies to circumsporozoite (CS) protein within one year (in the absence of re-exposure). Three patients had high levels of CS antibodies even after one year, and this persistence was related to the level of the initial antibody response. In the area of the outbreak 10% of schoolchildren had antibodies to the (NANP) 40 peptide. 21% of the 42 children with present or past overt malaria were antibody positive. Of the children with no such background, 8% were antibody positive. The corresponding seropositivity rates for asexual blood stages were 31% and 1% for the 2 groups respectively. It is concluded that (NANP) 40 ELISA is potentially a valuable tool in sero-epidemiology, particularly in situations of seasonal transmission and recurrences due to drug resistance.

Partial characterization of a rapidly released antigenic/allergenic component (Ag 5) of Aspergillus fumigatus

An antigenic component of Aspergillus fumigatus, which was responsible for inducing an initial early antibody response in rabbits immunized with A. fumigatus germlings, has been identified and partially characterized. By immunofluorescent antibody and ELISA techniques, this antigen was demonstrated to be present on the germling surface, although it was also detected in supernatants of conidia/germlings within 1 hour and was present in all shake and stationary culture-filtrate extracts. By incorporation of a monospecific antiserum to this component in an intermediate gel of the reference self-crossed radioimmunoelectrophoresis pattern for allergic bronchopulmonary aspergillosis sera, it was recognized as Ag 5 and was also demonstrated to bind specific IgE. Further immunochemical analyses have revealed that Ag 5 is relatively heat labile, does not bind to concanavalin A, and has a molecular weight of approximately 35 kd. This rapidly released antigenic/allergenic component may play an important role in the initiation of immunologic responses in patients with allergic bronchopulmonary aspergillosis.