Revisiting the Principles of Designing a Vaccine.

Immune principles formulated by Jenner, Pasteur, and early immunologists served as fundamental propositions for vaccine discovery against many dreadful pathogens. However, decisive success in the form of an efficacious vaccine still eludes for diseases such as tuberculosis, leishmaniasis, and trypanosomiasis. Several antileishmanial vaccine trials have been undertaken in past decades incorporating live, attenuated, killed, or subunit vaccination, but the goal remains unmet. In light of the above facts, we have to reassess the principles of vaccination by dissecting factors associated with the hosts' immune response. This chapter discusses the pathogen-associated perturbations at various junctures during the generation of the immune response which inhibits antigenic processing, presentation, or remodels memory T cell repertoire. This can lead to ineffective priming or inappropriate activation of memory T cells during challenge infection. Thus, despite a protective primary response, vaccine failure can occur due to altered immune environments in the presence of pathogens.

Development of the Antileishmanial Vaccine.

Search for an efficacious antileishmanial vaccine has led to clinical trials of numerous vaccine candidates in the past few decades. As no promising candidate has emerged from these studies, novel vaccine modalities and vaccine assessment techniques are still emerging for antileishmanial vaccine development. Briefly, this chapter discusses: (a) history and timeline of antileishmanial vaccine development; (b) techniques utilized for developing whole-parasite and subunit-based antileishmanial vaccine formulations, and (c) immunogenicity and post-challenge protective efficacy assessment of vaccine candidates.

LmjF.36.3850, a novel hypothetical Leishmania major protein, contributes to the infection.

Leishmania is a protozoan parasite that resides in mammalian macrophages and inflicts the disease known as leishmaniasis. Although prevalent in 88 countries, an anti-leishmanial vaccine remains elusive. While comparing the virulent and avirulent L. major transcriptomes by microarray, PCR and functional analyses for identifying a novel virulence-associated gene, we identified LmjF.36.3850, a hypothetical protein significantly less expressed in the avirulent parasite and without any known function. Motif search revealed that LmjF.36.3850 protein shared phosphorylation sites and other structural features with sucrose non-fermenting protein (Snf7) that shuttles virulence factors. LmjF.36.3850 was predicted to bind diacylglycerol (DAG) with energy value similar to PKCα and PKCß, to which DAG is a cofactor. Indeed, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue, enhanced the phosphorylation of PKCα and PKCßI. We cloned LmjF.36.3850 gene in a mammalian expression vector and primed susceptible BALB/c mice followed by challenge infection. We observed a higher parasite load, comparable antibody response and higher anti-inflammatory cytokines such as IL-4 and IL-10, while expression of major anti-leishmanial cytokine, IFN-γ, remained unchanged in LmjF.36.3850-vaccinated mice. CSA restimulated LN cells from vaccinated mice after challenge infection secreted comparable IL-4 and IL-10 but reduced IFN-γ, as compared to controls. These observations suggest a skewed Th2 response, diminished IFN-γ secreting Th1-TEM cells and increased central and effector memory subtype of Th2, Th17 and Treg cells in the vaccinated mice. These data indicate that LmjF.36.3850 is a plausible virulence factor that enhances disease-promoting response, possibly by interfering with PKC activation and by eliciting disease-promoting T cells.

Residue-Specific Message Encoding in CD40-Ligand.

CD40-Ligand (CD40L)-CD40 interaction regulates immune responses against pathogens, autoantigens, and tumor and transplantation antigens. Single amino acid mutations within the 115-155 amino acids stretch, which is responsible for CD40L functions, result in XIgM syndrome. We hypothesize that each of these amino acids of CD40L encodes specific message that, when decoded by CD40 signaling, induces a specific profile of functions. We observed that every single substitution in the XIgM-related amino acids in the 115-155 41-mer peptide in CD40L selectively altered CD40 signaling and effector functions-cytokine productions, HMGCoA reductase, ceramide synthase, inducible nitric oxide synthase and arginase expression, survival of B cells, and control of Leishmania infection and anti-leishmanial T cell response-suggesting residue-specific encoding of a distinct set of messages that collectively define CD40L pleiotropy, serve as a target for engineering the ligand to generate superagonists as immunotherapeutic, and implicate the evolutionary diversification of functions among the ligands in a protein superfamily.

Leishmania major adenylate kinase immunization offers partial protection to a susceptible host.

Leishmania major causes mild-to-severe cutaneous lesions resulting in significant disfigurations, if untreated. The drugs are toxic, and drug-resistance parasites are emerging. Therefore, a prophylactic vaccination is an urgent need. As no vaccine is available, we compared the genes expressed by virulent and avirulent parasites. We identify L major adenylate kinase (AdeK) as a probable vaccine candidate after a series of experimentations. We cloned the gene in mammalian pcDNA6/HisA and pet28a+ vector for in vivo expression following immunization and in vitro protein expression for booster, respectively. We observed that immunization of susceptible BALB/c mice with AdeK resulted in significant protection against L major challenge infection. The protection was accompanied by increased IFN-γ producing lymphocytes and reduced IL-4, IL-17 and IL-10 secreting central and effector Th2, Th17 and Treg memory cells, respectively. These observations indicate L major AdeK as a potential vaccine candidate.

LmjMAPK10 offers protection against Leishmania donovani infection.

AIMS: This study aimed at evaluating the DNA vaccination efficacy of Leishmania major-derived MAPK10 against Leishmania donovani infection. METHODS AND RESULTS: MAPK10 is one of the 15 mitogen-activated protein kinases (MAPKs) of Leishmania major. Herein, we expressed the gene through a mammalian vector and tested whether priming with this gene would offer protection against L donovani infection. We report that LmjMAPK10 DNA vaccination using a mammalian expression vector significantly reduces the parasite burden. The protection is accompanied by host-protective T-cell functions, TH 1-type cytokines and elevated leishmanial antigen-specific IgG2a isotype response. T-cell response to the L donovani/challenge infection is associated with increase in IL-12 and IFN-γ, but reduced IL-10 and IL-4 production. CONCLUSIONS: LmjMAPK10 is cross-protective against L donovani infection.

Anti-Leishmanial Vaccines: Assumptions, Approaches, and Annulments.

Leishmaniasis is a neglected protozoan parasitic disease that occurs in 88 countries but a vaccine is unavailable. Vaccination with live, killed, attenuated (physically or genetically) Leishmania have met with limited success, while peptide-, protein-, or DNA-based vaccines showed promise only in animal models. Here, we critically assess several technical issues in vaccination and expectation of a host-protective immune response. Several studies showed that antigen presentation during priming and triggering of the same cells in infected condition are not comparable. Altered proteolytic processing, antigen presentation, protease-susceptible sites, and intracellular expression of pathogenic proteins during Leishmania infection may vary dominant epitope selection, MHC-II/peptide affinity, and may deter the reactivation of desired antigen-specific T cells generated during priming. The robustness of the memory T cells and their functions remains a concern. Presentation of the antigens by Leishmania-infected macrophages to antigen-specific memory T cells may lead to change in the T cells' functional phenotype or anergy or apoptosis. Although cells may be activated, the peptides generated during infection may be different and cross-reactive to the priming peptides. Such altered peptide ligands may lead to suppression of otherwise active antigen-specific T cells. We critically assess these different immunological issues that led to the non-availability of a vaccine for human use.

Prevalence and identification of extended spectrum ß-lactamases (ESBL) in Escherichia coli isolated from a tertiary care hospital in North-East India.

Extended-spectrum ß-lactamases (ESBLs) are rapidly evolving group of ß-lactamase enzymes produced by the Gram negative bacteria. In this study, we determined the antimicrobial sensitivity pattern of Escherichia coli isolates and prevalence of TEM, SHV and CTX-M genes in ESBL positive E. coli isolated from the patients admitted to a tertiary care hospital in North-East India. A total of 85 multidrug-resistant isolates of E. coli obtained from clinical samples; urine (n = 80), sputum (n = 3), body fluid (n = 1), vaginal discharge (n = 1) were screened for resistance to third generation cephalosporins. ESBL production in resistant isolates was determined by double disk synergy test (DDST) and phenotypic confirmatory test (PCT). ESBL positive isolates were subjected to PCR for detection of TEM, SHV and CTX-M genes. Imipenem was found to be most effective against E. coli (susceptible isolates 96.47%) while ciprofloxacin was the least effective antibiotic (resistant isolates 60%). Among 33 ESBL positive isolates confirmed via PCT, preponderance in female population (60.6%) was noted. The most prevalent gene was bla(SHV) (63.04%) followed by bla(TEM) and bla(CTX-M) (60.86 and 54.34%, respectively) in ESBL positive E. coli. Most of the extensively used antibiotics, appear to be ineffective against the ever-mutating bacteria. This resistance urges cautious antimicrobial management on priority. Further, it helps in effectively designing the chemotherapeutic regimen for patients of a particular geographic area.

Identification of potential vaccine candidates against Streptococcus pneumoniae by reverse vaccinology approach.

In the past few decades, genome-based approaches have contributed significantly to vaccine development. Our aim was to identify the most conserved and immunogenic antigens of Streptococcus pneumoniae, which can be potential vaccine candidates in the future. BLASTn was done to identify the most conserved antigens. PSORTb 3.0.2 was run to predict the subcellular localization of the proteins. B cell epitope prediction was done for the immunogenicity testing. Finally, BLASTp was done for verifying the extent of similarity to human proteome to exclude the possibility of autoimmunity. Proteins failing to comply with the set parameters were filtered at each step. Based on the above criteria, out of the initial 22 pneumococcal proteins selected for screening, pavB and pullulanase were the most promising candidate proteins.

Vancomycin-resistant enterococci: Troublemaker of the 21st century.

The emergence of multidrug-resistant and vancomycin-resistant enterococci during the last decade has made it difficult to treat nosocomial infections. Although various enterococcal species have been identified, only two (Enterococcus faecalis and Enterococcus faecium) are responsible for the majority of human infections. Vancomycin is an important therapeutic alternative against multidrug-resistant enterococci but is associated with a poor prognosis. Resistance to vancomycin dramatically reduces the therapeutic options for enterococcal infections. The bacterium develops resistance by modifying the C-terminal d-alanine of peptidoglycan to d-lactate, creating a d-Ala-d-Lac sequence that effectively reduces the affinity of vancomycin for the peptidoglycan by 1000-fold. Moreover, the resistance genes can be transferred from enterococci to Staphylococcus aureus, thereby posing a threat to patient safety and also a challenge for treating physicians. Judicious use of vancomycin and broad-spectrum antibiotics must be implemented, but strict infection control measures must also be followed to prevent nosocomial transmission of these organisms. Furthermore, improvements in clinical practice, rotation of antibiotics, herbal drugs, nanoantibiotics and the development of newer antibiotics based on a pharmacogenomic approach may prove helpful to overcome dreadful vancomycin-resistant enterococcal infections.