Anamnestic Antibody Response Research Articles

Rabies vaccination: comparison of neutralizing antibody responses after priming and boosting with different combinations of DNA, inactivated virus, or recombinant vaccinia virus vaccines

Long-term levels of neutralizing antibody were evaluated in mice after a single immunization with experimental DNA or recombinant vaccinia virus (RVV) vaccines encoding the rabies virus glycoprotein (G), or the commercially available inactivated virus human diploid cell vaccine (HDCV). Anamnestic antibody titers were also evaluated after two booster immunizations with vaccines that were identical to or different from the priming vaccine. Five hundred and forty days (1.5 year) after a single immunization with any of the three vaccines, neutralizing antibody titers remained greater than the minimal acceptable human level of antibody titer (0.5 International Units (IU)/ml). In addition, either an HDCV or DNA booster elicited early and elevated anamnestic antibody responses in mice that had been primed with any of the three vaccines. In contrast, RVV boosters failed to elevate titers in mice that had been previously primed with RVV, and elicited slowly rising titers in mice that had been primed with either DNA or HDCV. Thus, a single vaccination with any of the three different vaccines elicited long-term levels of neutralizing antibody that exceeded 0.5 IU/ml. In contrast, different prime–booster vaccine combinations elicited anamnestic neutralizing antibody responses that increased quickly, increased slowly or failed to increase.

Recombinant modified vaccinia virus ankara expressing the surface gp120 of simian immunodeficiency virus (SIV) primes for a rapid neutralizing antibody response to SIV infection in macaques.

Neutralizing antibodies were assessed before and after intravenous challenge with pathogenic SIVsmE660 in rhesus macaques that had been immunized with recombinant modified vaccinia virus Ankara expressing one or more simian immunodeficiency virus gene products (MVA-SIV). Animals received either MVA-gag-pol, MVA-env, MVA-gag-pol-env, or nonrecombinant MVA. Although no animals were completely protected from infection with SIV, animals immunized with recombinant MVA-SIV vaccines had lower virus loads and prolonged survival relative to control animals that received nonrecombinant MVA (I. Ourmanov et al., J. Virol. 74:2740-2751, 2000). Titers of neutralizing antibodies measured with the vaccine strain SIVsmH-4 were low in the MVA-env and MVA-gag-pol-env groups of animals and were undetectable in the MVA-gag-pol and nonrecombinant MVA groups of animals on the day of challenge (4 weeks after final immunization). Titers of SIVsmH-4-neutralizing antibodies remained unchanged 1 week later but increased approximately 100-fold 2 weeks postchallenge in the MVA-env and MVA-gag-pol-env groups while the titers remained low or undetectable in the MVA-gag-pol and nonrecombinant MVA groups. This anamnestic neutralizing antibody response was also detected with T-cell-line-adapted stocks of SIVmac251 and SIV/DeltaB670 but not with SIVmac239, as this latter virus resisted neutralization. Most animals in each group had high titers of SIVsmH-4-neutralizing antibodies 8 weeks postchallenge. Titers of neutralizing antibodies were low or undetectable until about 12 weeks of infection in all groups of animals and showed little or no evidence of an anamnestic response when measured with SIVsmE660. The results indicate that recombinant MVA is a promising vector to use to prime for an anamnestic neutralizing antibody response following infection with primate lentiviruses that cause AIDS. However, the Env component of the present vaccine needs improvement in order to target a broad spectrum of viral variants, including those that resemble primary isolates.

An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients.

We describe the development of a capture enzyme-linked immunosorbent assay for the detection of the dengue virus nonstructural protein NS1. The assay employs rabbit polyclonal and monoclonal antibodies as the capture and detection antibodies, respectively. Immunoaffinity-purified NS1 derived from dengue 2 virus-infected cells was used as a standard to establish a detection sensitivity of approximately 4 ng/ml for an assay employing monoclonal antibodies recognizing a dengue 2 serotype-specific epitope. A number of serotype cross-reactive monoclonal antibodies were also shown to be suitable probes for the detection of NS1 expressed by the remaining three dengue virus serotypes. Examination of clinical samples demonstrated that the assay was able to detect NS1 with minimal interference from serum components at the test dilutions routinely used, suggesting that it could form the basis of a useful additional diagnostic test for dengue virus infection. Furthermore, quantitation of NS1 levels in patient sera may prove to be a valuable surrogate marker for viremia. Surprisingly high levels of NS1, as much as 15 microg/ml, were found in acute-phase sera taken from some of the patients experiencing serologically confirmed dengue 2 virus secondary infections but was not detected in the convalescent sera of these patients. In contrast, NS1 could not be detected in either acute-phase or convalescent serum samples taken from patients with serologically confirmed primary infection. The presence of high levels of secreted NS1 in the sera of patients experiencing secondary dengue virus infections, and in the context of an anamnestic antibody response, suggests that NS1 may contribute significantly to the formation of the circulating immune complexes that are suspected to play an important role in the pathogenesis of severe dengue disease.

Management of sickle cell disease: recent advances and controversies.

Management of sickle cell disease: recent advances and controversies.

Accelerated clearance of SHIV in rhesus monkeys by virus-like particle vaccines is dependent on induction of neutralizing antibodies

Recombinant, insect cell derived SIV Pr56 gag virus-like particles (VLPs) have been modified either by inserting HIV-1 Gp160 derived peptides into the Pr56 gag precursor or by integrating the complete HIV-1 gp120 in the particle membrane. To investigate the protective efficacy of these particulate antigens, rhesus macaques were immunized with VLPs both adjuvant-free or adsorbed to alum. In addition, recombinant Semliki Forest viruses (SFV) expressing proteins corresponding to the VLP constructs were established and administered as live vaccines in combination with particulate antigens. Vaccination induced specific humoral responses irrespective of the immunization regimen. However, in contrast to Pr56 gag-specific antibodies, Env-specific antibody titers could not be increased by booster immunizations in this study. Cell-mediated immune responses were detected following vaccination with VLP-preparations as well as recombinant SFVs. A tendency towards stimulating both enhanced cell mediated as well as humoral immune responses was observed following priming with recombinant SFVs. Upon challenge with SHIV-4 all vaccinated monkeys became infected. However, animals, that were vaccinated with VLPs presenting the complete gp120, managed to clear virus faster than nonimmunized controls. The observed virus elimination significantly correlated with an anamnestic antibody response and an accelerated appearance of neutralizing antibodies postchallenge.

Immunogenicity of various presentation forms of PorA outer membrane protein of Neisseria meningitidis in mice

In this study we compare different vaccine formulations containing meningococcal PorA outer membrane protein; purified PorA, outer membrane vesicles (OMV) and immune-stimulating complexes (iscom). Bactericidal antibodies could be generated by the OMV and iscom formulation but not with purified PorA using either AlPO 4 or Quil-A as adjuvant. OMV and iscom formulations revealed similar immunogenicity when tested in a dose response manner, with respect to bactericidal as well as OMV-binding antibodies. The anti-OMV IgG subclass response induced by PorA in OMV formulation was found in all subclasses IgG1, IgG2a, IgG2b, IgG3. OMP–iscoms induced very high IgG1 anti-OMV antibodies but almost no IgG3 response. Also, OMP–iscoms appeared to be a potent inducer of antibodies directed against linear peptides corresponding to surface exposed loops of PorA. In addition, iscoms as well as purified PorA with Quil-A as adjuvant (but not with AlPO 4) induced high levels of antibodies against purified PorA. In summary, in addition to the OMV formulation, only iscoms containing PorA are able to generate an anamnestic and bactericidal antibody response.

Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles.

The recognition that rotaviruses are the major cause of life-threatening diarrheal disease and significant morbidity in young children has focused efforts on disease prevention and control of these viruses. Although the correlates of protection in children remain unclear, some studies indicate that serotype-specific antibody is important. Based on this premise, current live attenuated reassortant rotavirus vaccines include the four predominant serotypes of virus. We are evaluating subunit rotavirus vaccines, 2/6/7-VLPs and 2/4/6/7-VLPs, that contain only a single VP7 of serotype G1 or G3. In mice immunized parenterally twice, G3 virus-like particles (VLPs) induced a homotypic, whereas G1 VLPs induced a homotypic and heterotypic (G3) serum neutralizing immune response. Administration of three doses of G1 or G3 VLPs induced serum antibodies that neutralized five of seven different serotype test viruses. The inclusion of VP4 in the VLPs was not essential for the induction of heterotypic neutralizing antibody in mice. To confirm these results in another species, rabbits were immunized parenterally with two doses of 2/4/6/7-VLPs containing a G3 or G1 VP7, sequentially with G3 VLPs followed by G1 (G3/G1) VLPs, or with live or psoralen-inactivated SA11. High-titer homotypic serum neutralizing antibody was induced in all rabbits, and low-level heterotypic neutralizing antibody was induced in a subset of rabbits. The rabbits immunized with the G1 or G3/G1 VLPs in QS-21 were challenged orally with live G3 ALA rotavirus. Protection levels were similar in rabbits immunized with homotypic G3 2/4/6/7-VLPs, heterotypic G1 2/4/6/7-VLPs, or G3/G1 2/4/6/7-VLPs. Therefore, G1 2/4/6/7-VLPs can induce protective immunity against a live heterotypic rotavirus challenge in an adjuvant with potential use in humans. Following challenge, broad serum heterotypic neutralizing antibody responses were detected in rabbits parenterally immunized with G1, G3/G1, or G3 VLPs but not with SA11. Immunization with VLPs may provide sufficient priming of the immune system to induce protective anamnestic heterotypic neutralizing antibody responses upon subsequent rotavirus infection. Therefore, a limited number of serotypes of VLPs may be sufficient to provide a broadly protective subunit vaccine.

Immune response to rabies booster vaccination in subjects who had postexposure treatment more than 5 years previously.

Patients exposed to rabies, who had a history of having received a complete series of intramuscular or intradermal postexposure vaccine treatment 5 or more years previously, all developed an early anamnestic antibody response to two intradermal booster vaccine injections. I t is suggested that such subjects do not require a full five-dose postexposure series nor do they require administration of rabies immune globulin. These data confirm World Health Organization and US-CDC recommendations. Current World Health Organization (WHO) recommendations state that indi&duals,previously vaccinated against rabies with a potent tissue or avian culture vaccine, should be given two tissue or avian culture vaccine boosters on days 0 and 3 ifpostexposure vaccination is required. Boosters can be ahnis te red either intramuscularly as a full ampoule or i n t r a d e r d y as 0.1 mL if the ampoule is reconstituted in 0.5 mL solution or 0.2 mL. if the diluent provided with the vaccine is 1 .O mL.'** Previous studies supported this recommendation.' Investigators at this institution published a study of nerve tissue-derived vaccine recipients, reexposed to rabies up to 50 years after their original immunization, and found that most but not all had an accelerated immune response and thus showed immunological memory. This was not related to the time elapsed since the previous Semple or

Characterization of CD4+ T cells in mouse bone marrow. I. Increased activated/memory phenotype and altered TCR Vbeta repertoire.

A significant proportion of memory B cells home to bone marrow (BM) which is a major site of anamnestic antibody responses in mice. We hypothesized that memory T cells likewise accumulate in BM perhaps to provide help for antibody production, and that the compartment of CD4+ T cells in BM of unimmunized mice would be enriched for memory phenotype cells that might have been activated by environmental antigens. The phenotype of activated/memory CD4+ lymphocytes has been defined as CD44hi CD45RBlo CD62L-. Conversely, the phenotype of immunologically naive cells is CD44lo CD45RBhi CD62L+. Flow cytrometric analysis of tissue from normal, adult C57BL/6 mice identified 1-2 % CD3+CD4+ cells in BM. Up to 40 % of CD3+CD4+ cells in the BM expressed the activated/memory phenotype compared with < or = 10% in the spleen and lymph nodes. Analysis of TCR Vbeta repertoire revealed that expression of Vbeta3 and Vbeta7 genes was increased as much as fourfold in BM compared to the periphery; most of this increase was within the CD44hi T cells. The accumulation of activated/memory T cells and clonotypic expansion(s) was not seen in the BM of germ-free mice, indicating that it reflects the history of the animal's exposure to antigens. Finally, immunization of mice which express a transgenic T cell receptor specific for ovalbumin peptide resulted in appearance of antigen-specific T cells with activated/memory phenotype in the BM.

Analysis of the original antigenic sin antibody response to the major outer membrane protein of Chlamydia trachomatis.

The anamnestic antibody response to the Chlamydia trachomatis major outer membrane protein (MOMP) was evaluated in mice after priming with serovar C and boosting either with the homologous serovar or with heterologous serovars (A, H, K, and B). Microimmunofluorescence antibody responses demonstrated that boosting with heterologous serovars strongly recalled antibody to serovar C, typical of an original antigenic sin (OAS) response. Boosting with serovars antigenically related to serovar C (A, H, and K) recalled antibody to the variable domain 1 (VD1) peptide of the MOMP of serovar C as determined by a pin-peptide ELISA. Complete amino acid substitution analysis of the VD1 peptide epitope of the MOMP showed that the original antigenic sin response to each boosting serovar contained antibodies with unique patterns of VD1 peptide recognition. The data suggest that antigenically related C. trachomatis serovars differentially recruit B cell lineages from a heterogeneous memory B cell pool that had been induced by priming with the original serovar and thus account for the OAS antibody response.

CD4(+) T-lymphocyte and immunoglobulin G2 responses in calves immunized with Anaplasma marginale outer membranes and protected against homologous challenge.

Protective immunity against the ehrlichial pathogen Anaplasma marginale has been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain of A. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both whole A. marginale homogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4(+) T cells, and CD4(+) T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response to A. marginale and produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinct A. marginale strains, and Anaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes against A. marginale challenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.

DNA immunization protects nonhuman primates against rabies virus.

More than 40,000 people die annually from rabies worldwide. Most of these fatalities occur in developing countries, where rabies is endemic, public health resources are inadequate and there is limited access to preventive treatment. Because of the high cost of vaccines derived from cell culture, many countries still use vaccines produced in sheep, goat or suckling mouse brain. The stability and low cost for mass production of DNA vaccines would make them ideal for use in developing countries. To investigate the potential of DNA vaccines for rabies immunization in humans, we vaccinated Macaca fascicularis (Cynomolgus) monkeys with DNA encoding the glycoprotein of the challenge virus standard rabies virus, or with a human diploid cell vaccine (HDCV). The monkeys then were challenged with a non-passaged rabies virus. DNA or HDCV vaccination elicited comparable primary and anamnestic neutralizing antibody responses. All ten vaccinated monkeys (DNA or HDCV) survived a rabies virus challenge, whereas monkeys vaccinated with only the DNA vector developed rabies. Furthermore, serum samples from DNA- or HDCV-vaccinated monkeys neutralized a global spectrum of rabies virus variants in vitro. This study shows that DNA immunization elicits protective immunity in nonhuman primates against lethal challenge with a human viral pathogen of the central nervous system. Our findings indicate that DNA vaccines may have a promising future in human rabies immunization.

Definition of T cell epitopes within the 19 kDa carboxylterminal fragment of Plasmodium yoelii merozoite surface protein 1 (MSP1(19)) and their role in immunity to malaria.

MSP1(19) is one of the leading malaria vaccine candidates. However, the mechanism of protection is not clear. To determine whether MSP1(19)-specific effector T cells can control parasitaemia, we analysed the specificity of T cells induced following immunization with recombinant forms of P. yoelii MSP1(19) and asked whether they could protect mice. There was no evidence that effector T cells were capable of protecting since: (1) immunization of mice with yMSP1(19), but not defined epitopes, was able to induce protection; and (2) long term MSP1(19)-specific CD4+ T cell lines were incapable of adoptively transferring protection. In contrast, priming mice with the T cell epitopes resulted in a rapid anamnestic antibody response to MSP1(19) after either challenge with MSP1(19) or parasite. Thus, MSP1(19) contains multiple T cell epitopes but such epitopes are the targets of helper T cells for antibody response but not of identified effector T cells capable of controlling parasitaemia.

Detection of antibodies to Candida albicans germ tubes during experimental infections by different Candida species.

Identification and characterization of Candida albicans germ tube-specific antigens may be of relevance for the serodiagnosis of invasive candidiasis since they could be the basis for the development of new diagnostic tests. In this study, we have identified two antigens of 180 and >200 kDa in the cell wall of C. albicans germ tubes which are responsible for the induction of antibodies to C. albicans germ tubes. Antigens of similar molecular masses have been demonstrated in the cell walls of the Candida species C. stellatoidea, C. parapsilosis, C. guilliermondii, C. tropicalis, and C. krusei, but not C. glabrata. The kinetics of the antibody responses to C. albicans germ tubes were studied in rabbits infected with different Candida species. Although these antibodies were detected in rabbits infected with all Candida species except C. glabrata, the kinetics of the antibody responses to C. albicans germ tubes induced by the Candida species studied were different. Both the highest titer and the earliest response of antibodies to C. albicans germ tubes were observed in rabbits infected with either of the two serotypes of C. albicans used. However, the time needed to elicit the antibodies to C. albicans germ tubes can be reduced as the result of an anamnestic antibody response. The results presented in this study show that a test designed to detect antibodies against C. albicans germ tube antigens may be suitable for the diagnosis of infections caused by most of the medically important Candida species.

A two-dose hepatitis B vaccine regimen: proof of priming and memory responses in young adults

This study shows that two doses of a recombinant hepatitis B vaccine (10 μg or 20 μg of HBsAg per dose), administered 6 months apart to young, healthy adults, can induce an antibody (anti-HBs) response similar to that expected with the standard three-dose regimen of this vaccine given at intervals of 0, 1, and 6 months. While only 46–67% of the vaccinees displayed a protective anti-HBs titer of ≥10 mIU ml −1 prior to the receipt of the second dose at 6 months, virtually all were primed as 97–99% of the subjects developed such a titer when tested a month after the second dose. Among vaccinees given 10 or 20 μg doses, respectively, the secondary rise in antibody following the second dose yielded geometric mean titers (GMTs) of 1103 and 2538 mIU ml −1, respectively. The study further demonstrated that a two-dose regimen of vaccination induced strong immunologic memory for HBsAg, as a booster dose of vaccine given 2 years later resulted in a rapid and vigorous anamnestic antibody response.

Successful nucleic acid based immunization of newborn chimpanzees against hepatitis B virus

To determine whether DNA based immunization could protect newborn chimpanzees against a challenge infection with hepatitis B virus, two chimpanzees were immunized on the day of birth with a plasmid coding for hepatitis B surface antigen, and boosted at 6 and 24 weeks. Both animals produced transient antibody to the hepatitis B surface antigen. Following challenge with hepatitis B virus at 33 weeks the two immunized animals developed anamnestic antibody responses, however, neither developed detectable hepatitis B surface antigen or antibody to the core protein, the conventional markers of hepatitis B infection. Both of these markers appeared in an unimmunized control animal. We conclude that DNA based immunization can induce protective immunity in newborn chimpanzees.

History of Immunology: Paul Ehrlich: The Founding of Pediatric Immunology

In 1892–93, Paul Ehrlich published a series of experiments on the passive transfer of maternal antibody to fetus and newborn that deserve to be better known. He pointed out the importance of mother's milk to neonatal well-being. He was the first to define the difference between active and passive immunity. He was the first to demonstrate immune elimination of antigen and to explain the significance of each phase of the kinetics of the anamnestic antibody response. We point out how elegant were these early experiments and how far in advance of their times.

Fine specificity of equine infectious anaemia virus gp90-specific antibodies associated with protective and enhancing immune responses in experimentally infected and immunized ponies.

Equine infectious anaemia virus (EIAV) provides a model for examining the natural immunological control of a persistent lentivirus infection and for evaluating the efficacy of various vaccine strategies. As an initial characterization of antibody responses associated with protective or enhancing immune responses elicited by experimental infections or vaccinations, we have utilized synthetic peptide ELISA to characterize the fine specificity of antibodies to linear determinants of the EIAV surface glycoprotein, gp90. The data indicated that serum antibodies associated with protective or enhancing immune responses differed quantitatively and qualitatively in their pattern of reactivity to gp90 peptides. Protective and enhancing EIAV vaccines could also be distinguished by their ability to evoke anamnestic antibody responses to gp90 peptides. These studies demonstrate for the first time definitive differences in the specificity of protective and enhancing antibody responses to EIAV and emphasize the importance of using native viral glycoprotein immunogens in lentivirus vaccines.

Long-Term Antibody Responses of Cats Fed Toxoplasma gondii Tissue Cysts

As part of a long-term study on immunity to oocyst shedding, 12 4-6-mo-old cats were inoculated orally with tissue cysts of the ME-49 strain (6 cats) or the TS-2 strain (6 cats) of Toxoplasma gondii. Two cats fed the ME-49 strain died or were killed because of acute toxoplasmosis 12 and 13 days after inoculation (DAI), respectively. On day 39 after primary infection, 5 cats (2 infected with the ME-49 strain and 3 infected with the TS-2 strain) were challenged orally with tissue cysts of the ME-49 strain. One cat died following rechallenge infection due to causes unrelated to toxoplasmosis. Seventy-seven months after primary infection, the remaining 9 cats were challenged orally with tissue cysts of the P89 strain of T. gondii. Blood samples were obtained weekly or monthly and sera were analyzed for antibodies to T. gondii using the modified agglutination test (MAT), the Sabin-Feldman dye test (DT), and the enzyme-linked immunosorbent assay (ELISA) for IgM (IgM-ELISA) or IgG (IgG-ELISA). The MAT was performed using both formalin-fixed (FF) and acetone-fixed (AF) tachyzoites. The MAT (FF) was the most sensitive test; cats seroconverted within 14 DAI and high titers (> 10,000) persisted > 6 yr, although cats had no clinical signs. The MAT titers using the AF detected recent exposure and titers declined sharply after 2 mo postinoculation. DT and ELISA titers were lower and developed slower than MAT titers. Fluctuations in antibody titers were limited to 8-fold during the 6-yr observation period. Anamnestic serum antibody responses were seen in 2 cats after the final challenge, but not after first challenge.

Absence of Protective Resistance to Homologous Challenge Infections in Jirds with Chronic, Amicrofilaremic Infections of Brugia pahangi

Most jirds (Meriones unguiculatus) with chronic Brugia pahangi infections remain microfilaremic, develop a hyporesponsive state, and are susceptible to reinfection. Although few, some jirds become amicrofilaremic or fail to develop a microfilaremia. The hypothesis that chronically infected, amicrofilaremic jirds may be resistant to reinfection was tested. Twenty-four chronically infected amicrofilaremic jirds, with or without circulating antigen, were inoculated subcutaneously with 75 B. pahangi third-stage larvae (L3). Necropsies were performed 25 days postinoculation, and challenge populations were separated from existing worms by size. Similar inoculations and necropsies were performed on groups of chronically infected microfilaremic jirds and uninfected jirds. Based on worm recoveries, jirds with occult infections were not resistant to reinfection. An anamnestic antibody response to extracts of L3 or microfilariae (mf) was not seen, nor were antibodies to the surface of L3 or mf. The data indicate that a susceptible state is maintained in this model of lymphatic filariasis in the absence of circulating mf.