Ayan Dey

Enzyme-linked immunospot assays for direct ex vivo measurement of vaccine-induced human humoral immune responses in blood

The enzyme-linked immunospot (ELISPOT) assay was originally developed to enumerate antigen-specific antibody-secreting cells (ASCs), and has subsequently been adapted for various applications, including the detection cytokine-secreting cells. Owing to its exceptionally high sensitivity, the ELISPOT has proven to be especially useful for detecting discrete populations of active cells (e.g., antigen-specific cells). Because of its versatility, the ELISPOT assay is used for a wide range of applications, including clonal analyses of immune responses after vaccination or after immunotherapy. Here we describe standard protocols for the detection of human ASCs specific to virtually any vaccine antigen after enrichment of circulating plasmablasts. In addition, a protocol is described for the measurement of mucosal ASC responses after prior immunomagnetic enrichment of mucosally derived blood lymphocytes. The protocols described allow rapid (∼6–8 h) detection of specific ASCs in small (1–2 ml) samples of blood and can be performed in resource-poor settings.

Applications of molecular methods for Leishmania control

This article reviews the recent advances made in the field of human leishmaniasis. Special emphasis is placed upon the application of various molecular tools for accurate and rapid diagnosis, understanding the mechanisms of drug resistance and identification of vaccine candidates. The focus will be on the major role played by recombinant antigens in the immunoserodiagnosis and progress of the Leishmania genome project, which has enabled researchers to design better PCR primers and molecular probes for microarrays. A special interest is placed on the recombinant antigen (rK39) cloned from the Leishmania chagasi kinesin gene and a very recently cloned recombinant antigen (KE16) from the Old World Leishmania donovani species with high sensitivity and specificity. Advances made in the specific PCR primer designed to diagnose and differentiate various species and strains of Leishmania causing visceral and post-kala-azar-dermal leishmaniasis have been covered. Molecular methods (e.g., DNA and protein microarrays) applied to understanding the pathobiology of the parasite, mechanism of host invasion, drug interaction and drug resistance to develop effective therapeutic molecules, gene expression profiling studies that have opened doors to understand many host–parasite relations, effective therapy and vaccine candidates are extensively covered in this review.

Emerging Leptospirosis, North India

To the Editor: We read with interest the article, The Changing Epidemiology of Leptospirosis in Israel, published in volume 7, no. 6 (1). Leptospirosis, a septicemic zoonosis with multisystemic involvement, is caused by the pathogenic strains of Leptospira interrogans. Rural farm workers are at high risk for leptospirosis, and it can be a significant public health problem when water and food safety are not ensured. Several epidemics of leptospirosis have occurred on Andaman and Nicobar islands and in southern and western parts of India during the past century (2). The organism has been detected in farm animals in many parts of the country (3); however, human infections have been more or less localized. In 1998, researchers warned that, unless adequate public health measures were initiated, large leptospirosis epidemics were possible in areas where the disease had not been previously reported (4). In addition, they recommended improving clinical diagnosis and conducting systematic epidemiologic studies to control of the disease (4). The true incidence of human leptospirosis in northern India is not known either because of a lack of awareness on the part of the treating physicians or the lack of diagnostic techniques. In 1966, human leptospirosis was reported in Delhi, a state in northern India (5). In a 1966 study (5), sera from persons with pyrexia and jaundice were tested by the agglutination lysis test for leptospiral antibodies. Of 93 serum specimens from persons with pyrexia cases, 3 were positive (1 with L. icterohemorrhagica and two with L. canicola); of 43 serum specimens from persons with jaundice, 3 were positive (2 with L. icterohemorrhagica and 1 with L. icterohemorrhagica and L. pomona). No other study on leptospirosis has been done in the region, and no data are available concerning the problem. To assess the current status of transmission in Delhi and its adjoining areas, we conducted a systematic study for the diagnosis of leptospirosis in our hospital from April 2000 to March 2001; case definition criteria suggested in a previous study (4) were used. A case was defined as a person with fever, headache, and myalgia and more than two of the following symptoms: jaundice, oliguria, respiratory symptoms (cough, hemoptysis, and breathlessness), hemorrhagic manifestations (hematemesis, bleeding gums, and subconjunctival hemorrhage), and signs of meningeal irritation and convulsion. Seventy-five patients (44 male patients; 3–73 years of age) satisfied the inclusion criteria. In addition to clinical evaluation and assessment for other diseases, leptospirosis was investigated by the following laboratory methods: isolation of Leptospira interrogans, direct visualization of the organism under dark-field microscopy, and enzyme-linked immunosorbent assay (ELISA) for Leptospira immunoglobulin (Ig) M antibody (Serion Immunodiagnostica GmbH, Wurzburg, Germany). Per manufacturer’s specifications, the sensitivity, specificity, positive predictive value, and negative predictive value of this kit are 96%, 97%, 90%, and 99%, respectively). All blood samples were sent to the Leptospira referral laboratory at the Indian Veterinary Research Institute, Izzatnagar, for microscopic agglutination test (MAT). Eight serovars of L. interrogans (australis, autumnalis, pomona, sejroe, tarassovi, icterohaemorrhagica, hebdomadis, and patoc) were tested, and a agglutination titer of more than 1:100 was considered positive. All patients were treated empirically with broad-spectrum antibiotics as well as specific drugs according to the results of investigations. Thirty-two patients (42.6%) had a positive ELISA test for Leptospira IgM antibody. The results of MAT were positive in 21 (65.6%) of the 32 ELISA-positive serum samples. Serum specimens from 11 patients reacted with a single serovar, and specimens from 10 patients reacted with more than one serovar. Among the pathogenic species, Leptospira antibodies were detectable by MAT predominantly against L. sejroe (7 of 21), followed by L. icterohaemorrhagica (6 of 21), L. hebdomadis (4 of 21), and L. tarassovi (4 of 21). Leptospira antibodies were also detectable against L. autumnalis (3 of 21), L. australis (2 of 21), and L. pomona (1 of 21). Against L. patoc, MAT could detect antibodies in six samples. The organism could not be isolated in culture or visualized under dark-field microscopy in any of the specimens. Of the 43 case-patients with ELISA-negative specimens, alternative diagnoses were established for 40 on the basis of various laboratory investigations. In five of the case-patients with ELISA-positive specimens, coinfection with other pathogens was detected, including Salmonella typhi (one case) by a positive Widal test, hepatitis C virus by positive ELISA (two cases), and Plasmodium falciparum (two cases) by a positive smear. Five patients, including three who were ELISA positive for Leptospira, died. The highest number of ELISA-positive serum samples (21 of 32) were obtained in August and September 2000, suggesting an epidemic. Epidemiologic investigation of leptospirosis is often hampered by the difficulty of making a definitive microbiologic diagnosis. Isolation of leptospira from clinical samples provides a definitive diagnosis; however, the value of culture is limited because samples have to be collected before the administration of antibiotics, and culturing requires prolonged incubation. Demonstration of typical motility of leptospira under dark-ground illumination in clinical samples, though helpful in early diagnosis, has low sensitivity and depends on the technician’s opinion. Measurement of IgM antibodies against Leptospira by ELISA has emerged as a reliable diagnostic test with good specificity and sensitivity (6). The probability of achieving a positive serologic test increases with the duration of disease, and good correlation between results of MAT and ELISA has been reported by Cumberland et al. (7). MAT has emerged as a dependable diagnostic tool for leptospirosis (next to isolation) by providing serovar specific diagnosis. However, a large number of serovars of L. interrogans exist, and maintaining large numbers of organisms for MAT is difficult for most laboratories. Moreover, MAT may fail to detect antibodies when specific serovars are not used. In this study, the ELISA-positive samples, for which MAT results were negative, may have been caused by infection with serovars other than those used in this study. Because of the problems with methods, leptospirosis is grossly underdiagnosed. Leptospira organisms require humid weather for their survival. Rodents and domestic animals (i.e., cattle and dogs) harbor leptospires and shed the bacteria in urine; they may disseminate the organism in the rain and drinking water sources. Humans frequently come into contact with contaminated water during floods; the number of cases is higher during and after heavy rainfalls. We found that the peak incidence of the disease was during August and September, the monsoon season, which may explain the high incidence of seropositivity during this period. Though the organism has been detected in farm animals in northern India, human leptospirosis has not been considered a major public health problem, probably because transmission is low in arid weather conditions. As a result of 13 consecutive monsoons of above-average strength in India, changes in the environment may be promoting the transmission of this organism. Recently, two other regions in northern India, Chandigarh (8) and Varanasi (9), have reported a Leptospira seroprevalance rate of 8.8% and 21.74%, respectively. Our study supports the warning from other researchers regarding the threat of leptospirosis in areas such as northern India. Preventive measures should be initiated and rapid and definitive diagnostic tests must be developed.

Expression and characterization of a recombinant kinesin antigen from an old Indian strain (DD8) of Leishmania donovani and comparing it with a commercially available antigen from a newly isolated (KE16) strain of L. donovani

Recently we had prepared a recombinant antigen (Ld-rKE16) from a newly isolated Indian strain of Leishmania donovani (MHOM/IN/KE16/1998) with high sensitivity and specificity and the same has been commercialized. While comparing the sequence data of kinesin gene of this (KE16) strain and its expressed protein with another commercially available recombinant antigen (Lc-rK39) from kinesin gene of L. chagasi we found significant genetic and amino acid variations. This prompted us to undertake the present study to unravel whether the kinesin gene and its expressed protein from another old but Indian isolate of L. donovani (MHOM/IN/DD8/1968) had any genetic and amino acid heterogeneity. Sequencing of the kinesin gene revealed that the kinesin gene of DD8 strain is 3016bp long and has immunodominant region consisting of 4.8 tandem repeats, 117 base pairs each. Further blast analysis of the immunodominant regions of 5 strains of L. donovani revealed that it has only 79% homology with L. chagasi, and 80% homology with L. infantum; while it had 82% homology with Sudan strain of L. donovani, 82% with another (Morena) strain of Indian L. donovani but highest homology of 83% with L. donovani KE16 strain of India. We also evaluated the diagnostic potential of the recombinant DD8 antigen (Ld-rDD8) and compared the results with that of Ld-rKE16. The study revealed that Ld-rKDD8 antigen was less sensitive and specific as compared to rKE16 antigen for the diagnosis of visceral and post-kala-azar dermal leishmaniasis. This was probably due to prolong in vitro culture maintenance of the DD8 strain.

Kinesin motor domain of Leishmania donovani as a future vaccine candidate.

ABSTRACT Visceral leishmaniasis (VL) is one of the important parasitic diseases, with approximately 350 million people at risk. Due to the nonavailability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. The present study was carried out to examine the immunological potential of kinesin protein from the microtubule locus of Leishmania donovani as a suitable vaccine candidate. In silico analysis of this region revealed clusters of major histocompatibility complex class I and II binding epitopes in its motor domain region. A recombinant protein was expressed from this region and named rLvacc. The antigenicity and immunogenicity studies of this protein by Western blot analysis revealed that rLvacc is strongly recognized by sera from acute VL patients. To evaluate its immunogenicity, peripheral blood mononuclear cells from cured VL patients were separated, and a lymphocyte proliferation assay was carried out in the presence of rLvacc. After lymphocyte proliferation, the pooled culture supernatant was assayed for anti-rLvacc antibody titers using an enzyme-linked immunosorbent assay. The results showed that immunoglobulin G2 (IgG2) subtype antibodies were predominant, while IgG1 subtype antibodies were produced in very low titers. On the basis of these ex vivo preliminary findings, its immunogenicity was studied in BALB/c mice. Vaccination with the DNA construct generated a good cellular immune response with significant increases in gamma interferon and interleukin-2 (IL-2) cytokine levels (Th1), but no increase in IL-4 levels (Th2). Taken together, our findings suggest the kinesin motor domain region of L. donovani as a potential vaccine candidate against visceral leishmaniasis.

Use of Immunoglobulin G Avidity To Determine the Course of Disease in Visceral and Post-Kala-Azar Dermal Leishmaniasis Patients

ABSTRACT In the present study, anti-Leishmania immunoglobulin G (IgG) avidity was used to estimate the approximate time of disease manifestation. Significant differences (P < 0.0001) were found between the levels of anti-rKE-16 IgG avidity in leishmaniasis patients with recent and chronic diseases. More than 76% of patients with an illness duration of less than 6 months had avidity of less than 70%, 94% of patients had less than 80% avidity, and all (100%) patients with illness of more than 6 months had avidity values higher than 70%. The study showed that avidity could successfully be used to pinpoint the duration of leishmaniasis.

A bivalent conjugate vaccine containing PspA families 1 and 2 has the potential to protect against a wide range of Streptococcus pneumoniae strains and Salmonella Typhi.

Previously we showed that conjugation of pneumococcal surface protein A (PspA) to Vi capsular polysaccharide from Salmonella Typhi enhanced the anti-PspA response without the need to add adjuvant. In the current study conjugates consisting of the α helical regions of PspA families 1 or 2 bound to Vi were used to vaccinate mice to test their ability to protect against a lethal intravenous challenge of a range of various strains of Streptococcus pneumoniae. Conjugate vaccine containing PspA family 1 provided good protection from PspA family 1 challenge strains but offered very little protection against PspA family 2 challenge strains. Similarly, PspA family 2 conjugates provided good protection from PspA family 2 challenge strains and poor protection against PspA family 1 challenge strains. This observation was supported by the low levels of cross-reactivity of PspA antibodies seen in ELISA plates coated with the heterologous PspA family. Cytokine profiles showed a mixed Th1/Th2 response to Vi and the Vi-PspA conjugates. IgG subclass analysis of the anti-Vi response showed a shift from predominantly IgG2a/3 to IgG1 after conjugation to PspA was consistent with other polysaccharide conjugate vaccines. The results demonstrate that conjugation of the α helical region of PspA to Vi enhances its capacity to induce a protective immune response and that a vaccine based on the α helical region of PspA should contain PspA from both families 1 and 2 to achieve broad cross-protection.

Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus

Background The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine. Methods 199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation. Results An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4β7+ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7+ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7+ IgA- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus. Discussion Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to evaluate the duration of such memory responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the polio eradication program.

Immunogenicity of candidate chimeric DNA vaccine against tuberculosis and leishmaniasis

Mycobacterium tuberculosis and Leishmania donovani are important intracellular pathogens, especially in Indian context. In India and other South East Asian countries, both these infections are highly endemic and in about 20% cases co-infection of these pathogens is reported. For both these pathogens cell mediated immunity plays most important role. The available treatment of these infections is either prolonged or cumbersome or it is ineffective in controlling the outbreaks and spread. Therefore, potentiation of a common host defense mechanism can be used to prevent both the infections simultaneously. In this study we have developed a novel chimeric DNA vaccine candidate comprising the esat-6 gene of M. tuberculosis and kinesin motor domain gene of L. donovani. After developing this novel chimera, its immunogenicity was studied in mouse model. The immune response was compared with individual constructs of esat-6 and kinesin motor domain. The results showed that immunization with chimeric DNA vaccine construct resulted in stronger IFN-gamma and IL-2 response against kinesin (3012+/-102 and 367.5+/-8.92pg/ml) and ESAT-6 (1334+/-46.5 and 245.1+/-7.72pg/ml) in comparison to the individual vaccine constructs. The reciprocal immune response (IFN-gamma and IL-2) against individual construct was lower (kinesin motor domain: 1788+/-36.48 and 341.8+/-9.801pg/ml and ESAT-6: 867.0+/-47.23 and 170.8+/-4.578pg/ml, respectively). The results also suggest that using the chimeric construct both proteins yielded a reciprocal adjuvant affect over each other as the IFN-gamma production against chimera vaccination is statistically significant (p<0.0001) than individual construct vaccination. From this pilot study we could envisage that the chimeric DNA vaccine construct may offer an attractive strategy in controlling co-infection of leishmaniasis and tuberculosis and have important implication in future vaccine design.

Shigella outer membrane protein PSSP-1 is broadly protective against Shigella infection.

ABSTRACT In developing countries, Shigella is a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multiple Shigella serotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigella surface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection against Shigella flexneri serotypes 2a, 5a, and 6, Shigella boydii, Shigella sonnei, and Shigella dysenteriae serotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conserved Shigella protein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine against Shigella.