Quantitation of immunoglobulin classes and subclasses in anti-Rh (D) antibodies

The isotype profiles of anti-Rh (D) antibodies were measured from 16 serum samples with a solid-phase radioimmunoassay that employed monoclonal anti-isotype antibodies. These antibodies had been standardized in another solid-phase system with the aid of monoisotypic antibodies (e.g., anti-tetanus toxoid of isotype IgG4), and this permitted calculations of the relative concentrations of different isotypes in each anti-D population. Of the heavy-chain isotypes, only IgG1 and IgG3 were found in anti-Rh (D) antibodies. The share of IgG1 varied from 100% (2 sera) to 0% (1 serum) of detected units, and the mean proportion was 73.8%. The remainder was IgG3 (mean share 26.2%). The proportions of kappa and lambda immunoglobulins varied greatly in individual sera, in three sera only kappa Ig and in one serum only lambda Ig could be detected.     

Antibody response to a purified parasite proteinase (SMw32) in Schistosoma mansoni infected mice

Indirect immunofluorescence of Schistosoma mansoni adult worm sections has revealed that the early immunoglobulin response is directed toward the parasite digestive tract. One of the components of the worm gut is a cysteine proteinase which degrades host hemoglobin ingested by the parasite. In this report the purified proteinase (SMw32) was used in ELISA and immunoblot analyses to study the specific antibody response during the course of an acute infection. We have found high titer IgG antibody in S. mansoni infected, but not uninfected, mice. The anti-proteinase response involves IgM, IgG1, IgG2a, and IgE isotypes. Total IgM and IgG levels increased by week 3 post-infection and remained elevated throughout the study (7 weeks). Increased titers (IgM, IgG) of specific anti-proteinase were also apparent by week 3 post-infection, long before fecal eggs were detectable. Mean anti-proteinase IgG stabilized at high titer by week 5 post-infection, while IgM titers decreased to near background levels. Anti-proteinase IgE was first detectable at week 4 and reached peak titers by weeks 6 and 7. The strong antibody response to the purified SMw32 proteinase is consistent with the early reactivity of S. mansoni infected mice and humans to a 31 kDa component of the worm gut described by others.     

Beneficial effects of the anti-oestrogen tamoxifen on systemic lupus erythematosus of (NZBxNZW)F1 female mice are associated with specific reduction of IgG3 autoantibodies

BACKGROUND: Sex hormones have been shown to influence the immune system and to modify the course of autoimmune disorders. OBJECTIVE: To examine the effects of the oestrogen antagonist tamoxifen on the course of systemic lupus erythematosus (SLE) in (NZBxNZW)F1 mice. METHODS: Groups of 8 week old (NZBxNZW)F1 female mice were treated with tamoxifen (800 micro g/mouse; twice a week) or with double distilled water for four months. Mice were evaluated monthly for the presence of autoantibodies directed against DNA and nuclear extract (NE) by enzyme linked immunosorbent assay (ELISA). White blood cells and thrombocytes were quantified by a cell counter and proteinuria by combistix kit. At 6 months of age, all mice that did not die spontaneously were killed and evaluated for the presence of glomerular immune deposits by indirect immunofluorescence assay. IgG isotypes of autoantibodies in the mouse sera and glomeruli were determined by gamma chain specific antibodies. RESULTS: Tamoxifen treatment significantly reduced autoantibody production directed against either NE or DNA. The latter reduction was mainly in autoantibodies of the IgG3 isotype. Furthermore, tamoxifen had significant beneficial effects on the course of SLE in (NZBxNZW)F1 mice. At 6 months of age, 40% of the untreated mice died spontaneously, whereas all the tamoxifen treated mice were still alive. All untreated mice showed severe thrombocytopenia and persistent proteinuria, with diffuse glomerular immune deposits of IgG2a and IgG3 isotypes in their kidneys. In contrast, the tamoxifen treated mice had a normal number of thrombocytes and only minimal proteinuria. Moreover, glomerular immune deposits were detected in <40% of the tamoxifen treated mice. The latter were mainly of the IgG2a but not of the IgG3 isotype. CONCLUSION: The results clearly show the remarkable therapeutic effects of tamoxifen on SLE of (NZBxNZW)F1 female mice and suggest that these beneficial effects are related to the specific reduction of IgG3 autoantibodies.     

Antibody isotypes of immune complexes in schistosomiasis mansoni in Sudan

From a panel of monoclonal antibodies to Schistosoma mansoni, one that is specific to a shared cercarial and schistosomular antigen, and does not react with other major parasite species including Fasciola hepatica, was selected for use as an antigen-capture layer in a sandwich ELISA for detection of specific circulating immune complexes in the blood of S. mansoni-infected subjects from Sudan. The test, which identifies immune complexes of only a single antibody-bound antigen, is developed using human Ig class- and IgG subclass-specific enzyme (HRP)-antibody conjugates. European blood donor sera and those with rheumatoid factor and/or anti-nuclear antibody are negative in this test. The prevalence and distribution of the different antibody isotypes in antigen-specific complexes was determined in 276 subjects of four infection groups; primary school children, adult irrigation canal cleaners with chronic infections, an equivalent group of Praziquantel cured canal cleaners, and hospital-referred cases with severe hepatosplenic symptoms. The isotype profiles of antibodies in the specific complexes were compared with those in the total serum complexes prepared by polyethylene glycol precipitation. In chronic infections and in children there is a high prevalence of IgG and IgM specific complexes, the IgG being predominantly IgG1, with little or no IgG3 and IgG4. Treated chronic infections show reduced specific complexes in all classes of antibody. Compared with chronic and children's infections, a large proportion of the patients with severe infections have in addition to high IgM, high levels of IgG2, IgG3 and IgG4 specific complexes, a fact which suggests a causal relationship between antibody production in one or more of these subclasses to the circulating antigen and symptoms of hepatosplenic disease. Although all subjects have significant amounts of total serum complexes with IgG, untreated chronic infections have much higher concentrations than other infected groups. This group also has the highest levels and prevalence of IgM and IgE complexes. In treated chronic cases IgG and IgM total complexes are greatly reduced. The significant finding in patients with severe symptoms is the relatively high IgA immune complex level compared with other groups. Taken overall the results suggest that screening of populations in endemic regions for serum immune complexes by specific and non-specific means could offer valuable data on the significance of antibody responses to circulating antigens in different isotypes in relation to pathogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prophylactic effect of the anti-inflammatory drug diclofenac in experimental schistosomiasis mansoni.

OBJECTIVES: This study was a trial to demonstrate the prophylactic effect of diclofenac, a widely used anti-inflammatory drug (diclofenac potassium, CAS-15307-81-0, Ciba Geigy, 334.2) in experimental schistosomiasis mansoni. Two different dose regimens were used to explore the effects upon worm load, tissue egg load, and hepatic granuloma size. METHODS: In this study, a group of 50 Swiss albino mice was used. This group was divided into five subgroups: subgroup I constituted infected untreated control mice; subgroup II, infected mice given 0.5 mg diclofenac orally 24 h post infection, then sacrificed three weeks later; subgroup III, infected mice given 0.5 mg diclofenac orally six weeks post infection and sacrificed one week later; subgroup IV, infected mice administered 1mg diclofenac orally 24 h post infection and sacrificed three weeks later; and subgroup V, infected mice given 1mg of the drug orally six weeks post infection and sacrificed one week later. RESULTS: Mice given the high dose regimen (1mg orally/mouse) 24 h post infection, then sacrificed three weeks later, demonstrated a significant reduction in the immature worms recovered, compared to the untreated controls. Animals receiving the high dose of the drug six weeks post infection, then sacrificed one week later, revealed a drop in the number of mature worms and in the tissue egg load (hepatic and intestinal), and the smallest hepatic granuloma measurement compared to the untreated controls. These findings were less conspicuous in animals given the low dose regimen. CONCLUSION: Diclofenac could be used successfully as a preventive agent against schistosomiasis mansoni infection in endemic areas.     

Prevalence and clinical significance of isotype specific antinuclear antibodies in primary biliary cirrhosis

BACKGROUND: Antinuclear antibodies (ANA) giving a rim-like/membranous (RL/M) or a multiple nuclear dot (MND) pattern are highly specific for primary biliary cirrhosis (PBC).Aim and SUBJECTS: To assess the prevalence of PBC specific ANAs, their Ig isotype, and their clinical significance in 90 PBC patients from Greece and Spain. Twenty eight patients with chronic hepatitis C, 23 patients with systemic lupus erythematosus, and 17 healthy subjects were studied as controls. METHODS: PBC specific ANA reactivity was tested by indirect immunofluorescence using HEp2 cells as substrate and individual Ig class (IgG, IgA, IgM) and IgG subclass (IgG1, IgG2, IgG3, IgG4) specific antisera as revealing reagents. RESULTS: Fourteen of 90 (15.6%) PBC patients had PBC specific ANA reactivity when an anti-IgG (total) antiserum was used as the revealing reagent while 58 (64.4%) were positive when specific antisera to each of the four IgG isotypes were used. The prevailing isotype was IgG3 for MND and IgG1 for RL/M. PBC patients with specific ANA, in particular of the IgG3 isotype, had significantly more severe biochemical and histological disease compared with those who were seronegative. None of the controls was positive. CONCLUSIONS: Disease specific ANA are present in the majority of patients with PBC when investigated at the level of immunoglobulin isotype. PBC specific ANA, in particular of the IgG3 isotype, are associated with a more severe disease course, possibly reflecting the peculiar ability of this isotype to engage mediators of damage.     

Chemotherapy-induced,age-related changes in antischistosome antibody responses

Humoral responses directed against Schistosoma mansoni soluble egg antigen were studied in Zimbabwean children before and after treatment with either praziquantel (PZQ) or oxamniquine (OXAM). Treated children showed a significant increase in the proportion producing IgE and IgG3 and in mean levels of IgE, IgM, IgG3 six weeks post-treatment. At 18 weeks post-treatment, the proportion of treated children producing IgA, IgE, and IgG3 increased while the proportion producing IgG1 and IgG4 decreased. Mean levels of IgA, IgE, and IgG3 were higher than pre-treatment levels while levels of IgG1, IgG4 and IgM were lower. Statistical analyses showed that the magnitude of change in levels of IgE, IgM and IgG3 at 6 weeks post-treatment and of IgE, IgG3 and IgG4 at 18 weeks post-treatment was significantly greater in treated compared to untreated children, and there were no significant differences in immune responses between children treated with praziquantel and those treated with oxamniquine. The magnitude of change in IgE at 6 and 18 weeks, IgM at 6 weeks and IgG3 at 18 weeks post-treatment were significantly associated with age in treated but not in untreated children, with the change being greater in younger children. This suggests that treatment induced a change in the age-antibody relationship for these isotypes, and that the age-antibody relationship is not robust to chemotherapy. Pre-treatment infection levels were significantly associated (positive correlation) with the magnitude of change for IgE and IgG3 at 18 weeks post-treatment. Taken together, these results indicate that the age-antibody relationship observed in these children is due, at least in part, to cumulative host experience of parasite antigens and not host age alone.     

Lineage-restricted retention of a primitive immunoglobulin heavy chain isotype within the Dipnoi reveals an evolutionary paradox

The lineage leading to lungfishes is one of the few major jawed vertebrate groups in which Ig heavy chain isotype structure has not been investigated at the genetic level. In this study, we have characterized three different Ig heavy chain isotypes of the African lungfish, Protopterus aethiopicus, including an IgM-type heavy chain and short and long forms of non-IgM heavy chains. Northern blot analysis as well as patterns of V(H) utilization suggest that the IgM and non-IgM isotypes are likely encoded in separate loci. The two non-IgM isotypes identified in Protopterus share structural features with the short and long forms of IgX/W/NARC (referred to hereafter as IgW), which were previously considered to be restricted to the cartilaginous fish. It seems that the IgW isotype has a far broader phylogenetic distribution than considered originally and raises questions with regard to the origin and evolutionary divergence of IgM and IgW. Moreover, its absence in other gnathostome lineages implies paradoxically that the IgW-type genes were lost from teleost and tetrapod lineages.     

Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish

During the analysis of Ig superfamily members within the available rainbow trout (Oncorhynchus mykiss) EST gene index, we identified a unique Ig heavy-chain (IgH) isotype. cDNAs encoding this isotype are composed of a typical IgH leader sequence and a VDJ rearranged segment followed by four Ig superfamily C-1 domains represented as either membrane-bound or secretory versions. Because teleost fish were previously thought to encode and express only two IgH isotypes (IgM and IgD) for their humoral immune repertoire, we isolated all three cDNA isotypes from a single homozygous trout (OSU-142) to confirm that all three are indeed independent isotypes. Bioinformatic and phylogenetic analysis indicates that this previously undescribed divergent isotype is restricted to bony fish, thus we have named this isotype "IgT" (tau) for teleost fish. Genomic sequence analysis of an OSU-142 bacterial artificial chromosome (BAC) clone positive for all three IgH isotypes revealed that IgT utilizes the standard rainbow trout V(H) families, but surprisingly, the IgT isotype possesses its own exclusive set of D(H) and J(H) elements for the generation of diversity. The IgT D and J segments and tau constant (C) region genes are located upstream of the D and J elements for IgM, representing a genomic IgH architecture that has not been observed in any other vertebrate class. All three isotypes are primarily expressed in the spleen and pronephros (bone marrow equivalent), and ontogenically, expression of IgT is present 4 d before hatching in developing embryos.     

The relationship between age,sex, egg-count and specific antibody responses against Schistosoma mansoni antigens in a Ugandan fishing community

In schistosomiasis endemic areas, antibody isotype responses against Schistosoma mansoni antigens vary with host age, sex and duration or intensity of infection, and are associated with susceptibility or resistance to infection. Identifying the quality and quantity of these responses is important to our understanding of the host-parasite relationship; however, the various host and parasite factors have a strong tendency to confound each other. We investigated the relationships and interactions between age, sex, faecal egg-counts and specific antibody isotype (IgA, IgG1, IgG2, IgG3, IgG4, IgE, IgM) responses to S. mansoni worm (SWA) and egg (SEA) antigens, amongst 380 individuals aged 5-59 from a fishing community from Uganda. This community was characterized by high levels of exposure, and high infection intensities, with higher infection intensities in males than in females. Multivariate anova was conducted with interaction terms between the three categorized explanatory variables. Most anti-SWA responses increased with age, whereas anti-SEA responses tended to decline with age, especially after puberty. IgG1-SWA, IgG4-SWA, IgG4-SEA, IgE-SWA responses increased with egg count, whereas IgG2-SEA decreased with egg count. IgG1-SWA, IgG4-SWA, IgE-SWA and IgG4-SEA responses were independently higher in males, whereas IgG2-SEA responses were independently higher in females. The significant effects of sex on isotype responses to adult worm antigens may be partly because of different levels of cumulative exposure. IgG4-SEA and IgG4-SWA were both strongly correlated with egg count. Patterns of IgE-SWA responses were qualitatively different to IgG4 responses, suggesting independent pathways of regulation.     

Antibody response of Echinococcus granulosus infected mice: Protoscolex specific response during infection is associated with decreasing specific IgG1/IgG3 ratio as well as decreasing avidity

The antibody response was followed during 68 weeks in 17 Balb/c mice intraperitoneally (i.p.) infected with Echinococcus granulosus protoscoleces (PSC) and in three mice i.p. immunized with dead PSC. Titres of antibodies recognizing peptidic and glucidic PSC epitopes, as well as their isotypic and avidity profiles were followed by ELISA. In addition, antigen recognition patterns were analysed by immunoblot. The response against carbohydrate epitopes was dominant in infected and immunized mice but stronger in the first group. Infected mice showed similar profiles of specific IgG and IgM with maximum titres from week 38 to 53. Although IgG1 and IgG3 were the predominant antibody subclasses, the ratio of IgG1/IgG3 antibody titres as well as antibody avidity decreased during the experiment, encompassing a decrease in recognition of peptidic epitopes. Immunized mice did not show significant levels of specific IgM and, after week 15, showed IgG titres lower than the infected mice. IgG1 was the predominant IgG subclass during all the experiment with background levels of IgG3. The mean Ab avidity was high and showed no significant changes during immunization. Different patterns of response were thus produced by dead and developing live parasites. Although high avidity IgG1 antibodies were early found in both cases, lower avidity IgG3 antibodies were increasingly produced afterwards only in infected animals. The isotype switch and avidity decrease observed only during infection are consistent with a possible parasitic mechanism to evade host immunity .     

The Humoral Immune Response in Melioidosis Patients during Therapy

Background: This study was undertaken to identify and quantify the class and subclass antibody responses to the culture filtrate antigen (CFA) of Burkholderia pseudomallei in melioidosis patients under long-term maintenance or eradication therapy. Materials and Methods: Sequential sera samples from seven melioidosis patients collected between January 1992 and April 1998 were analyzed for immunoglobulin (Ig) types and IgG isotypes by ELISA using B. pseudomallei CFA. Results: Melioidosis patients generated a strong IgG, IgA and IgM response to the CFA of B. pseudomallei throughout the infection and IgG1 and IgG2 were the predominant IgG istotypes produced. Although high levels of these antibodies were detected in all the seven patients, the IgG, IgG1 and IgG2 antibodies showed a consistent response and good correlation with the clinical history in all cases. Conclusion: This study suggests that monitoring IgG antibody or IgG1 or IgG2 isotype antibody levels to CFA in patients under maintenance or eradication antibiotic therapy may be useful as a tool to detect the status of infection and as a guideline to determine the duration of maintenance antimicrobial therapy. Received: February 4, 2002 · Revision accepted: October 6, 2002 C. Vasu (corresponding author)     

Inbred mice infected with Plasmodium yoelii differ in their antimalarial immunoglobulin isotype response

Antibodies are known to be important in mediating malarial immunity, but the influence of the various immunoglobulin isotypes on parasite elimination is unclear. The purpose of this study was to provide basic information on the induction of isotype expression in genetically different mice during primary malaria. Parasitaemias and the serum antimalarial IgM, IgG1, IgG2, IgG3 and IgA antibody titres measured in a radioimmunoassay were followed in outbred and 11 inbred strains of mice infected with 17XNL Plasmodium yoelii. Severity of infection, as judged by length of infection, peak parasitaemias and death, was found to differ between the strains. All strains developed rapid IgM responses, but only 3/11 inbred strains produced significant antimalarial IgG1 levels during primary infection. All strains produced an IgG2 response, which developed slightly more quickly in strains with the least severe courses of malaria. A large variation in the IgG3 response was noted between strains. In general, IgG3 antibodies were the first IgG-isotype to appear in serum. They were detected as early as day 8 in strains that developed mild infections but were not present until around day 20 in strains with the most severe cases of malaria. Only one strain produced detectable antimalarial IgA antibodies. These results show that different patterns of isotype expression are induced in inbred strains of mice during primary P. yoelii infection.     

Egg-hatching inhibition in mice immunized with recombinant Schistosoma bovis 28 kDa glutathione S-transferase

The capacity of a recombinant glutathione S-transferase from Schistosoma bovis (rSb 28GST) to protect BALB/c mice against homologous and heterologous infections with, respectively, S. bovis or Schistosoma mansoni has been studied. Two injections of the rSb 28GST and an intravenous boost resulted in a marked specific IgG response on the day of experimental challenge with S. bovis or S. mansoni cercariae. Immunization of BALB/c mice led to a reduction in egg maturation and egg viability after infection with S. bovis or S. mansoni. Adult worm recoveries after an S. bovis challenge infection and tissue egg densities (intestine and liver) in S. mansoni challenge infection were also reduced in the immunized groups, but these differences were not statistically significant. No association between in vitro inhibition of GST enzymatic activity induced by immunized mouse sera and worm burden reduction was recorded. The analysis of the immune response, on the day of perfusion, showed the production of immunoglobulin (Ig)G1, IgG2a and IgG2b specific antibodies and the production of interleukin (IL)-4 and IL-5 by spleen cells after rSb 28GST stimulation. These data suggest that rSb 28GST immunization induces a moderate effect upon egg maturation and egg hatching, suggesting the involvement of similar mechanisms of action and common, but not exclusive, targets during S. bovis and S. mansoni infections. As a consequence, immunization with rSb 28GST may prove useful in affecting the pathology and transmission of African schistosomes.     

Antibodies to Schistosoma mansoni in human cerebrospinal fluid

Cerebrospinal fluid (CSF) and serum samples from patients suspected of having neuroschistosomiasis (NS) were evaluated by an enzyme-linked immunosorbent assay. Monoclonal antibodies of various immunoglobulin isotypes (IgM, IgA, IgE, total IgG, IgG1, IgG2, IgG3, and IgG4) were used to detect antibodies against Schistosoma mansoni soluble egg antigen (SEA) and soluble worm adult preparation (SWAP). Of the 83 CSF samples tested, 55% were reactive to SEA (26% were reactive only to SEA and 29% to both SEA and SWAP), 34% were reactive to SWAP (5% only to SWAP and 29% to both SEA and SWAP), and 40% were not reactive with any antigen. Cases that tested positive for SWAP in CSF and negative in serum were not found. Samples with high specific IgG antibody titers were selected for immunoglobulin isotype profiling. In the CSF samples, the antibodies against SEA and SWAP were mainly IgM, IgG1, and IgG4, although other immunoglobulins were also detected. Interestingly, nine patients had high levels of IgG1 only in the CSF. These results suggest that there is local synthesis of IgG1, and that this isotype could be an important immunologic marker in the diagnosis of NS.     

Effect of highly active antiretroviral therapy (HAART) on hypergammaglobulinemia in HIV infected patients

To investigate the mechanism of hypergammaglobulinemia in HIV infected patients, the effect of highly active antiretroviral therapy (HAART) on the hypergammaglobulinemia was analyzed. Involved in this study were 34 untreated, 21 HAART-effective (complete response) and 14 HAART-non-effective (partial response) patients. Serum levels of HIV-RNA and gammaglobulin and immunoglobulin (Ig) isotypes were measured. Mean HIV-RNA levels of untreated and partial response patients were 1.6 x 10(4) copies/ml and 0.4 x 10(4) copies/ml, respectively. HIV-RNA levels of all complete response patients were below 4.0 x 10(2) copies/ml. Mean gammaglobulin percentages of untreated, partial response and complete response patients were 24.4%, 21.8% and 17.9%, respectively (p < 0.01 in untreated vs complete response patients). Mean IgG levels in the three groups were 2,489 mg/dl, 1,947 mg/dl and 1,618 mg/dl, respectively (p < 0.001 in untreated vs complete response patients). IgA levels were high in some untreated patients and lower in complete response patients. IgE levels were increased in some untreated and partial response patients, but there was no significant difference among the three groups. These results suggested that the hypergammaglobulinemia found in HIV infected patients was associated with HIV replication. The activation mechanism might differ by Ig isotypes.     

Collagen-induced arthritis in an outbred group of rhesus monkeys comprising responder and nonresponder animals. Relationship between the course of arthritis and collagen-specific immunity

It is speculated that the autoimmune response to type II collagen (CII) is a driving force in the pathogenesis of human rheumatoid arthritis (RA). In this report, we describe the relationship between the induction of collagen arthritis and the CII-specific humoral, as well as cellular, immune response in rhesus monkeys. Ten of 14 monkeys immunized with bovine type II collagen (B-CII) developed polyarthritis. Susceptible animals showed a T cell response to B-CII; resistant animals did not. After the primary immunization, the humoral response to B-CII, as well as to rhesus monkey type II collagen, was dominated by antibodies of the IgM isotype in the susceptible animals and by antibodies of the IgG isotype in the resistant animals. Because of the close phylogenic relationship between the rhesus monkey and humans, these data contribute valuable information about the role of CII-specific immunity in the pathogenesis of human RA.     

Familial resemblance in humoral immune response to defined and crude Schistosoma mansoni antigens in an endemic area in Brazil.

This study addressed whether the humoral immune response to crude and defined Schistosoma mansoni antigens aggregates within families. The sample included 155 siblings from 42 nuclear families in Brazil. Sera examined by ELISA for antibody isotypes reactive to defined schistosome antigens and crude schistosome antigens (soluble adult worm antigen preparation and soluble egg antigen) demonstrated that there was a difference in sibling-pair correlations between defined and crude S. mansoni antigens. In contrast to the finding with crude antigens, egg-positive sibling pairs showed significant familial resemblance for all IgG subclasses and IgE to adult-stage antigens Smp20.8 and Smp50. Only the IgE and IgG4 isotypes showed familial resemblance to the egg-stage antigen, Smp40. Egg-negative sibling pairs showed significant familial resemblance only for IgE and IgG4 to Smp40. That both the IgE and IgG4 response to defined S. mansoni antigens showed familial resemblance is interesting in light of the converging evidence for the role of IgE and IgG4 in human susceptibility and resistance to reinfection.