Immunotherapy approach with recombinant survivin adjuvanted with alum and MIP suppresses tumor growth in murine model of breast cancer.

Survivin has received attention as a potential target for cancer immunotherapy because of its crucial role in oncogenesis. We undertook this study to evaluate the immunotherapeutic potential of combination of recombinant survivin along with adjuvant alum and immune modulator Mycobacterium indicus pranii (MIP). In vivo efficacy of the combination was studied in an invasive murine breast cancer model. Recombinant survivin protein was purified from Escherichia coli based expression system and characterized by western blotting. Purified survivin protein was combined with alum and MIP and was used for immunization of Balb/c mice. Antigen-primed animals were then challenged with syngeneic mammary tumor cells known as 4T-1. Balb/c mice spontaneously develop tumor when inoculated with 4T-1 cells. Antigen and adjuvant combination was immunogenic and significantly suppressed tumor growth in mice immunized with combination of recombinant survivin (10 µg), alum, and MIP. This is the first report that describes a combination immunotherapy approach using recombinant survivin, alum, and MIP in highly metastatic murine breast cancer model and holds promise for development of new biotherapeutics for cancer.

Combination immunotherapy with Survivin and luteinizing hormone-releasing hormone fusion protein in murine breast cancer model.

AIM: To investigate the therapeutic potential of two recombinant proteins, Survivin and luteinizing hormone-releasing hormone (LHRH) fusion protein [LHRH(6leu)-LTB] for immunotherapy of breast cancer. METHODS: Murine 4T-1 breast cancer model was used to evaluate the efficacy of recombinant proteins in vivo. Twenty four Balb/c mice were divided into 4 groups of 6 mice each. Recombinant Survivin and LHRH fusion protein, alone or in combination, were administered along with immunomodulator Mycobacterium indicus pranii (MIP) in Balb/c mice. Unimmunized or control group mice were administered with phosphate buffer saline. Each group was then challenged with syngeneic 4T-1 cells to induce the growth of breast tumor. Tumor growth was monitored to evaluate the efficacy of immune-response in preventing the growth of cancer cells. RESULTS: Preventive immunization with 20 µg recombinant Survivin and MIP was effective in suppressing growth of 4T-1 mouse model of breast cancer (P = 0.04) but 50 µg dose was ineffective in suppressing tumor growth. However, combination of Survivin and LHRH fusion protein was more effective in suppressing tumor growth (P = 0.02) as well as metastasis in vivo in comparison to LHRH fusion protein as vaccine antigen alone. CONCLUSION: Recombinant Survivin and MIP suppress tumor growth significantly. Combining LHRH fusion protein with Survivin and MIP enhances tumor suppressive effects marginally which provides evidence for recombinant Survivin and LHRH fusion protein as candidates for translating the combination cancer immunotherapy approaches.

Development of a novel recombinant LHRH fusion protein for therapy of androgen and estrogen dependent cancers.

LHRH based vaccines are promising candidates for therapy of androgen and estrogen dependent cancers. We report in this communication development of a novel recombinant protein vaccine candidate against LHRH. A synthetic gene was designed in which the codon sequence in the LHRH decapeptide was modified by substituting the codon for 6-glycine with that of l-leucine. Further the LHRH(6leu) gene was linked to heat-labile enterotoxin of E. coli (LTB) as carrier. This LHRH(6leu)-LTB gene was cloned into a prokaryotic expression vector under the control of inducible and strong bacteriophage T7 promoter to over-express LHRH(leu) fused to LTB as recombinant protein in E. coli. Recombinant LHRH(leu)-LTB protein of ∼14 kDa size, was purified from inclusion bodies using in-situ refolding on the column and Ni-NTA based immobilized affinity chromatography. Western blot confirmed the immunoreactivity of purified LHRH(leu)-LTB fusion protein with anti-LHRH monoclonal antibody. The vaccine protein was further characterized by mass spectroscopy, circular dichroism and fluorescence spectroscopy. This communication reports a recombinant LHRH fusion protein with potential for blocking of sex hormones production for eventual therapy of sex hormones dependent neoplasms.

Survivin: a unique target for tumor therapy.

Survivin is the smallest member of the Inhibitor of apoptosis (IAP) family of proteins, involved in inhibition of apoptosis and regulation of cell cycle. These functional attributes make Survivin a unique protein exhibiting divergent functions i.e. regulating cell proliferation and cell death. Expression pattern of Survivin is also distinctive; it is prominently expressed during embryonal development, absent in most normal, terminally differentiated tissues but upregulated in a variety of human cancers. Expression of Survivin in tumours correlates with not only inhibition of apoptosis and a decreased rate of cell death, but also resistance to chemotherapy and aggressiveness of tumours. Therefore, Survivin is an important target for cancer vaccines and therapeutics. Survivin has also been found to be prominently expressed on both human and embryonic stem cells and many somatic stem cell types indicating its yet unexplored role in stem cell generation and maintenance. Overall, Survivin emerges as a molecule with much wider role in cellular homeostasis. This review will discuss various aspects of Survivin biology and its role in regulation of apoptosis, cell division, chemo-resistance and tumour progression. Various molecular and immunotherapeutic approaches targeting Survivin will also be discussed.

A unique vaccine for control of fertility and therapy of advanced-stage terminal cancers ectopically expressing human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) appears soon after fertilization of the egg and plays a critical role in implantation of the embryo leading to the beginning of pregnancy. Vaccines developed against hCG prevent pregnancy without impairment of ovulation and disturbance of menstrual regularity. A new recombinant vaccine hCGß-LTB has been developed that is highly immunogenic in various strains of mice and intended for the control of fertility in women. An additional use of this vaccine is likely to be treatment of advanced-stage cancers that ectopically express hCG.

Making of a vaccine preventing pregnancy without impairment of ovulation and derangement of menstrual regularity and bleeding profiles.

The story of making a vaccine against human chorionic gonadotropin (hCG) for control of fertility is briefly reviewed. The choice of hCG was made on the consideration that it is not involved in the cascade of hormones leading to ovulation; hence, antibodies against hCG would neither disturb ovulation nor normal production of sex hormones by the female. It would not react with any other tissue of the body because no organ of a healthy noncancerous female expresses hCG. International Committee for Contraception Research played a historic role in testing its immunogenicity, safety and reversibility in women in Finland, Sweden, Chile and Brazil. The Population Council also conducted valuable long-term studies (5 years) in New York in 63 rhesus monkeys, which demonstrated the lack of pathological consequences of antibodies cross-reactive with species luteinizing hormone. The first-ever efficacy trials on a birth control vaccine established high efficacy (one pregnancy in 1224 cycles) of anti-hCG antibodies at and above 50 ng/mL titers. Fertility was regained in the immediate next cycle, at titers falling below 35 ng/mL. A recombinant vaccine, hCGß-LTB, has been made, which is highly immunogenic in mice. It is due to undergo toxicology studies prior to resumption of clinical trials. An additional utility of this vaccine is likely in advanced-stage terminal cancers expressing hCG/subunits.

Mycobacterium indicus pranii is a potent immunomodulator for a recombinant vaccine against human chorionic gonadotropin.

The objective of this work was to identify a human use-permissible adjuvant to enhance significantly the antibody response to a recombinant anti-hCG vaccine. Previous Phase II efficacy trials in sexually active women have demonstrated the prevention of pregnancy at hCG bioneutralization titers of 50ng/ml or more. Mycobacterium indicus pranii (MIP), a non-pathogenic Mycobacterium employed as an autoclaved suspension in aqueous buffer, significantly increased antibody titers in the FVB strain of mice. Three other genetic strains of mice: SJL, C3H, and C57Bl/6 responded with antibody titers several-fold higher than 50 ng/ml, which is the protective threshold in women, although there were differences in the peak titers attained. In addition, the duration of the antibody response was lengthened. The vaccine hCGß-LTB, given together with MIP, induces both a Th1 and Th2 response, which is reflected in the production of not only IgG1, but also a high proportion of IgG2a and IgG2b antibodies.

Development of a highly immunogenic recombinant candidate vaccine against human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) is synthesized soon after fertilization and is essential for embryonic implantation. A vaccine targeting hCG would be an ideal choice for immuno-contraception; an anti-hCG vaccine developed by Talwar et al., has previously undergone Phase II efficacy trials, providing proof of principle. These trials established the threshold levels of bio-neutralizing anti-hCG antibody titers required to prevent pregnancy; however, these titers (>50 ng/ml) were achieved in only 80% of immunized women. In this communication, we report a novel recombinant anti-hCG vaccine which demonstrates improved immunogenicity. hCGß was genetically fused at C-terminal to the B-subunit of E. coli heat-labile enterotoxin. The recombinant fusion protein (hCGß-LTB) was expressed in Pichia pastoris and, upon adsorption on Alhydrogel along with Mycobacterium indicus pranii (MIP) as an immuno-modulator, evoked a very high anti-hCG immune response in 100% of immunized BALB/c mice. This recombinant vaccine is expected to reduce cost as well as facilitate production of a molecularly consistent conjugate on a large scale.

Metabolic properties of lactobacilli in women experiencing recurring episodes of bacterial vaginosis with vaginal pH >or= 5.

While 60% of women experiencing recurring episodes of bacterial vaginosis (BV) with vaginal pH >or= 5 are depleted of resident probiotic lactobacilli, the remainder carry one or more strains of lactobacilli. Their ability to make D-lactic acid is, however, low (3.94 +/- 0.72 mM/L) compared to the D-lactic acid produced by strains from healthy vagina with vaginal pH approximately 4 (8.04 +/- 1.07 mM/L) culture supernatant of 0.5 McFarland concentration (P < 0.001).

Gonadotropin-releasing hormone/human chorionic gonadotropin beta based recombinant antibodies and vaccines.

Gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) are unique targets for the control of fertility. Immunological approaches to neutralizing these hormones have additional utility in cancer treatment. Vaccines have been developed against both GnRH and hCG and these have undergone Phase I/II clinical trials documenting their safety, reversibility and efficacy. The heterospecies dimer hCG vaccine prevented pregnancy in women of proven fertility without impairment of ovulation or derangement of menstrual regularity and bleeding profiles. The protective threshold of antibody titers to achieve efficacy was determined in these first-ever trials. Recently, a recombinant vaccine against the beta subunit of hCG linked to the B subunit of heat labile enterotoxin has been made and expressed as a glycosylated conjugate in Pichia pastoris. Experiments indicate its ability to generate antibodies above the protective threshold in all immunized Balb/c mice. Ectopic expression of hCG/hCGbeta is observed in many advanced stage cancers of various origins. A chimeric high affinity and specific recombinant antibody against hCGbeta linked to curcumin kills hCGbeta expressing T lymphoblastic leukemia cells without any deleterious effect. Several synthetic and recombinant vaccines have been developed against GnRH. These reduce serum testosterone to castration levels causing atrophy of the prostate. Three Phase I/II clinical trials conducted in India and Austria have shown that these vaccines elicit non-surgical reduction of testosterone, a fall in prostate specific antigen and clinical improvement of prostate carcinoma patients. A multimer recombinant vaccine against GnRH has high efficacy for sterilization of pigs and other animals.

Spectrum of Lactobacillus species present in healthy vagina of Indian women.

BACKGROUND & OBJECTIVE: Lactobacilli are depleted in vagina of women suffering from recurring episodes of bacterial vaginosis with vaginal pH >or=5. With the objective of making available probiotic lactobacilli for replenishment in such women, a study was undertaken to isolate and characterize the Lactobacilli present in women with eco-healthy vagina in Delhi. No information is so far available on the species of Lactobacilli resident in vagina of women in India. METHODS: Vaginal swabs were taken from 80 women with informed consent after ethical approval and grown in MRS broth. Gram-positive, catalase-negative bacilli generating about 200 bp amplicon by PCR with Lactobacillus genus specific primers were further characterized by employing species specific primers followed by sequencing of 16S rDNA. Isolates of the same species were differentiated by random amplified polymorphic DNA (RAPD) profiles. RESULTS: The predominant species isolated were L. reuteri present in 26 (32.5%) women, L. fermentum in 20 (25%), and L. salivarius in 13 (16.25%) women. Sequencing of 16S rDNA of 20 isolates showed that except for two isolates of L. plantarum, sequences of the remaining agreed well with PCR identification. None of the isolates had similar RAPD profile. INTERPRETATION & CONCLUSION: Our findings showed lactobacilli species present in healthy vagina of women in India differ from those reported from other countries. This information would be useful to development of probiotic tablets seeking to replenish the missing lactobacilli for reproductive health of women in India.

Elimination of high-risk human papillomavirus type HPV16 infection by 'Praneem' polyherbal tablet in women with early cervical intraepithelial lesions.

PURPOSE: 'Praneem', a polyherbal formulation developed by us, has successfully completed Phase II efficacy study for treatment of abnormal vaginal discharge due to reproductive tract infections that act as co-factors for HPV persistence. In the present study we evaluated potential anti-HPV activity of Praneem in women infected with high risk HPV type 16. METHODS: Twenty women molecularly diagnosed positive for HPV16 infection without or with low grade squamous intraepithelial lesion (LSIL) or inflammation were assigned to receive intra-vaginal, topical application of either Praneem tablet or placebo for 30 days excluding the days of menstrual period and were evaluated for persistence of HPV infection using HPV L1 consensus and HPV type 16-specific PCR as primary outcome. RESULTS: One course of Praneem treatment resulted in elimination of HPV in 6 out of 10 (60%) cases. A repeat treatment of four patients with persisting HPV infection resulted in clearance of HPV in two additional cases resulting in an overall 80% clearance of HPV 16 as against a spontaneous clearance of 10% (1/10) seen in the placebo arm. The elimination of HPV DNA was found to be accompanied by marked improvement in clinical symptoms and cytological abnormalities of Praneem-treated patients. CONCLUSION: Our results showed for the first time that a 30-day intra-vaginal application of the Praneem can result in elimination of HPV infection from the uterine cervix.

Selective killing of leukemia and lymphoma cells ectopically expressing hCGbeta by a conjugate of curcumin with an antibody against hCGbeta subunit.

OBJECTIVE: A variety of cancers ectopically express human chorionic gonadotropin beta (hCGbeta). Patients harboring such cancers have poor prognosis and adverse survival. A recombinant chimeric antibody, cPiPP, exhibiting high affinity and specificity for hCGbeta/hCG was engineered. This study was designed to determine whether this antibody alone or conjugated to curcumin can selectively kill tumor cells expressing hCGbeta. EXPERIMENTAL DESIGN: The study was carried out on MOLT-4 and U-937 cells expressing hCGbeta and on peripheral blood leukocytes of acute myeloid leukemia (AML) patients. The anticancerous compound curcumin was conjugated to cPiPP. The binding of cPiPP and cPiPP-curcumin conjugate to cells was studied by flow cytometry and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), FACS with propidium iodide staining, trypan blue exclusion assay and microscopy. RESULTS: The antibody did not impair the growth of MOLT-4 and U-937 cells in culture. Its conjugate with curcumin, however, was lethal to both cell lines. The immunoconjugate killed tumor cells bearing the CD33 marker of an AML patient expressing hCGbeta but did not have a similar action on cells of another AML patient with the CD13 marker but who was negative for hCGbeta. CONCLUSION: A humanized antibody against hCGbeta linked to curcumin has potential for therapy of hCGbeta-expressing tumors.

Phase II clinical trial with Praneem polyherbal tablets for assessment of their efficacy in symptomatic women with abnormal vaginal discharge (an ICMR task force study).

Abnormal vaginal discharge syndrome (AVDS) is a commonly observed gynaecological complaint for which women seek medical attention. The present study was conducted in six Indian Council of Medical Research centres with Praneem polyherbal tablets (PPT), to determine their efficacy in the treatment of symptomatic women with AVDS. Data are given on 141 subjects investigated. In total, 137 women (97%) reported complete (n=62, 44%) and partial (n=75, 53%) relief from symptoms after use of PPT for seven consecutive days. On speculum examination, 71 (74%) women were confirmed to be cured of AVDS. Microbiological tests could only be conducted microscopically for Trichomonas vaginalis, Candida albicans and bacterial vaginosis. It was observed that all women with T. vaginalis had this infection cured by PPT, and the cure rate was 77% for C. albicans and 68% for bacterial vaginosis. Seventy-eight women (55%) reported a transient burning sensation, mostly on the first 2 d of intake of PPT; however, they continued to use the tablets for the prescribed 7 d. This study lays the basis for an extended Phase II/III clinical trial, preferably randomized and comparing a larger number of women to confirm the safety and efficacy of PPT.

A partner monoclonal antibody to Moab 730 kills 100% of DU145 and PC3 androgen-independent cancer cells.

A number of therapeutic options are available for patients with prostate carcinoma till the time that the tumour is hormone dependent. However, no fully effective therapy is available for the treatment of androgen-independent prostate carcinomas. Antibodies directed at epitopes unique to or overexpressed on the cancer cells could be of therapeutic utility. A monoclonal antibody (Moab) 2C4 has been generated, which binds with cells of two androgenindependent prostate cancers, DU145 and PC3, and does not bind to peripheral blood leukocytes (PBLs) of healthy donors. This antibody, along with the previously developed Moab 730, kills 100% of both DU145 and PC3 cells in the presence of complement and does not have a deleterious effect on PBLs of healthy males. The anti-tumour action of the two antibodies prevents the establishment of DU145 cell tumour in nude mice in vivo. Moab 2C4 in combination with 730 has potential for use as therapy for androgen-independent cancers.

Potential of a novel polyherbal formulation BASANT for prevention of Chlamydia trachomatis infection.

The effect of a novel polyherbal formulation BASANT on Chlamydia trachomatis was studied. In vitro sensitivity testing was done by direct exposure of C. trachomatis (pre-infection incubation with BASANT) and exposure of C. trachomatis within HeLa 229 cells (post-infection incubation with BASANT). Pre-infection incubation of standard serovar D/UW-3/Cx with BASANT showed complete inhibition after 60, 30 and 15 min of incubation at concentrations of 12, 30 and 60 microg/mL, respectively. In the post-infection incubation, 8-10 microg/mL of BASANT showed complete inhibition of standard serovar D/UW-3/Cx as well as of five clinical isolates of C. trachomatis after 48 h of incubation. BASANT also inhibited a clinical isolate obtained from a doxycycline treatment failure patient at a concentration of 30 microg/mL. Both assays with standard and clinical isolates showed that BASANT has antimicrobial activity against C. trachomatis, suggesting the potential clinical utility of BASANT for the prevention of C. trachomatis infection by the sexual route.

A novel polyherbal microbicide with inhibitory effect on bacterial, fungal and viral genital pathogens.

A polyherbal cream (Basant) has been formulated using diferuloylmethane (curcumin), purified extracts of Emblica officinalis (Amla), purified saponins from Sapindus mukorossi, Aloe vera and rose water along with pharmacopoeially approved excipients and preservatives. Basant inhibits the growth of WHO strains and clinical isolates of Neisseria gonorrhoeae, including those resistant to penicillin, tetracycline, nalidixic acid and ciprofloxacin. It has pronounced inhibitory action against Candida glabrata, Candida albicans and Candida tropicalis isolated from women with vulvovaginal candidiasis, including three isolates resistant to azole drugs and amphotericin B. Basant displayed a high virucidal action against human immunodeficiency virus HIV-1NL4.3 in CEM-GFP reporter T and P4 (Hela-CD4-LTR-betaGal) cell lines with a 50% effective concentration (EC50) of 1:20000 dilution and nearly complete (98-99%) inhibition at 1:1000 dilution. It also prevented the entry of HIV-1(IIIB) virus into P4-CCR5 cells (EC50 approximately 1:2492). Two ingredients, Aloe and Amla, inhibited the transduction of human papillomavirus type 16 (HPV-16) pseudovirus in HeLa cells at concentrations far below those that are cytotoxic and those used in the formulation. Basant was found to be totally safe according to pre-clinical toxicology carried out on rabbit vagina after application for 7 consecutive days or twice daily for 3 weeks. Basant has the potential of regressing vulvovaginal candidiasis and preventing N. gonorrhoeae, HIV and HPV infections.

A polyherbal vaginal pessary with spermicidal and antimicrobial action: evaluation of its safety.

A polyherbal vaginal pessary (Praneem) has been formulated that has antimicrobial properties against genital pathogens in addition to spermicidal action. Thus, it has dual potential as a barrier method for contraception and for providing protection against some sexually transmitted infections. The present study reports the findings of a multicentre trial that was conducted to evaluate the safety of this product. Trials were carried out in 23 women in three centres in India: the Postgraduate Institute of Medical Education and Research, Chandigarh; Safdarjang Hospital, New Delhi; and Kamla Nehru Memorial Hospital, Allahabad. Thorough clinical and pelvic examinations were carried out as well as cervical cytology, blood biochemistry and haematology before and after use of the polyherbal pessary intravaginally once daily for 7 consecutive days. No toxicity was observed on clinical examination or by laboratory investigations. Daily intravaginal use of this pessary for 7 days had no adverse effects on cervical cytology or on metabolic and organ functions.