• Vaccine Ontology ID: VO_0000528
• Type: Conjugate vaccine
• Antigen: Arabinomannan (AM) oligosaccharides. They are derived from Lipoarabinomannan (LAM), a carbohydrate antigen expressed on the surface of mycobacteria which contains a manna polysaccharide core attached to a phosphatidyl inositol lipid moiety (Hamasur et al., 2003). The surface carbohydrate was hypothesized to yield CD1-specific immune responses, promote mycobacterial clearance, downregulate T cell proliferation, and interfere with activation of macrophages through interferon gamma intervention (Hamasur et al., 2003).
• Ag85B from M. tuberculosis H37Rv gene engineering:
  • Type: Conjugate protein purified from LAM
  • Description: The 28-kDa AM oligos were pooled and linked to tetanus toxoid (TT) or other M. tuberculosis proteins (Ag85B, 75-kDa protein) with thioether linkage generated by conjugation method. Conjugation consisted of amination of AMO with ammonium chloride and sodium cyanoborohydride, deasalting and treatment with 2-imminothiolane, and end conjugation between thiolated oligsaccharide derivatives with bromoacetylated proteins and tributyl phosphine (Hamasur et al., 2003).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000127
  • Description: Alum (aluminum hydroxide gel) adjuvant was used initially. Eurocine L3 suspension adjuvant was prepared using 1:1 ratio solid monooleate and oleic acid, Tris buffer with pH adjustment including NaOH, and subsequent sonification. Eurocine L3 emulsion was prepared using 1:1 ratio solid monooleate and oleic acid which were liquified prior to addition of soy bean oil (Hamasur et al., 2003).
• Preparation: Arabinomannan (AM) oligosaccharides derived from LAM of Mycobacterium tuberculosis H37Rv were isolated and covalently conjugated to tetanus toxoid (TT) or to short-term culture filtrate proteins (antigen 85B (Ag85B) or a 75kDa protein) from M. tuberculosis strain Harlingen (Hamasur et al., 2003).

• Tradename: BCG
• Vaccine Ontology ID: VO_0000771
• CDC CVX code: 19
• Type: Attenuated live vaccine
• Preparation: The evaluation of BCG lacks comparability among the vaccination studies due to the use of six different BCG sub-strains [Copenhagen-1331, Pasteur-1173P, Glaxo-1077, Tokyo-172, Russian, Moreau]. The comparative genomic analysis of different BCG strains has shown that there is no uniformity among all presently used vaccines and this genetic variability may contribute to the variation in protective efficacy seen in different geographic regions of the world (Gupta et al., 2007).
  
  In one case, Calmette et al. (1923) used decreasing doses (1 mg, .01 mg, and .002 mg) of tubercle bacilli to infect guinea pigs s.c. Bacterial loads increased in response to decreasing dose sizes. Similar results were acquired for later intramuscular and intracerebral infection procedures by other groups (SWEDBERG, 1951)
• Virulence: The original BCG strain lost its virulence after 39 passages by Albert Calmette and Camille Guerin, and was administered to humans i.d. on the 231st passage. The BCG bacteria showed altered colony morphologies and no virulence in experimental animals. BCG has shown a high degree of safety over the years (Ducati et al., 2006).
  
  --Calmette et al. used length of survival as a metric for virulence in their 1923 study (Agger et al., 2002).
  --The increased ability for M. bovis strains to retain virulence was noted by Lange in 1922 (SWEDBERG, 1951).
• Description: Albert Calmette and Camille Guerin, two French scientists, made an attenuated strain from another mycobacterium (M. bovis) by growing it for 13 years on culture. They observed a reduction in the virulence in animals through this period. When infants were given this vaccine, it provided a reduction in mortality by 90%. Since then, Bacille Calmette Guerin [BCG] has become the most widely used vaccine throughout the world. It is estimated that more than 3 billion people have received BCG. The protective efficacy of BCG vaccine against adult pulmonary TB has varied dramatically from 80 to 0% (Gupta et al., 2007).
  
  Development of the BCG strain was predated by numerous M. tuberculosis studies from the 1890s-1920s, including (Lote 1889, Strauss 1895, Moriya 1909, Romer 1903, Weber 1912, Binder 1915, and Browning and Gulbransen 1926) in a diverse set of hosts (including mouse, avian, human, bovine, and guinea pig hosts) (SWEDBERG, 1951).
  
  Several new subunit booster vaccinations are under development, including Ag85, Mtb72F, HspX and other DosR-controlled gene products, Rv3407 and other Rpf-controlled gene products, and heparin-binding hemagglutinin (HBHA) (Kaufmann, 2005).

Human Response

• Immune Response: TB Ag-specific mucosal, humoral and cellular immune response via T cell proliferation and production (Wang et al., 2009).
• Description: SL(E6-85B) vaccine, when combined with BCG vaccination, induced the strongest TB Ag-specific mucosal, humoral, and cellular immune responses comprised of increased proliferation of T cells, IFN-gamma expression, granzyme B production (Wang et al., 2009).
• Host Ifng (Interferon gamma) response
  • Description: The experimental data demonstrated that three time vaccinations with BCG in BALB/c mice induced significant TB Ag-specific IFN-gamma immune responses in splenocytes. Splenocytes were stimulated with the Ag85B protein 2 weeks after the final immunization (Wang et al., 2009).
  • Detailed Gene Information: Click Here.

Human Response

• Vaccination Protocol: DNA was obtained from the peripheral blood of 99 NMIBC patients who were prospectively randomized to receive postresection intravesical BCG or BCG with interferon alpha. (Chiong et al., 2011)
• Immune Response: Patients with the NRAMP1 D543N G:G genotype and allele 3 (GT)n polymorphism had decreased recurrence time after BCG therapy. (Chiong et al., 2011)
• Host GPX1 response
  • Description: Polymorphisms of the GPX1 gene may be associated with recurrence of BCa after BCG immunotherapy. (Chiong et al., 2011)
  • Detailed Gene Information: Click Here.
• Host SLC11A1 response
  • Description: Polymorphisms of the SLC11A1 genes may be associated with recurrence of BCa after BCG immunotherapy. (Chiong et al., 2011)
  • Detailed Gene Information: Click Here.

Human Response

• Vaccination Protocol: Intradermally vaccinated the infants within 48 hours of birth. Examination of whole blood stimulated ex vivo with 1.2×106 CFU BCG 10 weeks after newborn BCG vaccination of South African infants (Randhawa et al., 2011).
• Immune Response: Increased production of TH1-type T cell cytokines (Randhawa et al., 2011).
• Host GPX1 response
  • Description: After stimulation with TLR6 lipopeptide ligands, PBMCs from TLR6-deficient, BCG-vaccinated individuals secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR6 genotypes (Randhawa et al., 2011).
  • Detailed Gene Information: Click Here.
• Host human IFNG response
  • Description: Increased IFNG production associated with TLR1/6 presence 10 weeks post BCG vaccination (Randhawa et al., 2011).
  • Detailed Gene Information: Click Here.
• Host IL10 response
  • Description: IL10 expression post vaccination increased in presence of TLR1/6 (Randhawa et al., 2011).
  • Detailed Gene Information: Click Here.
• Host IL2 response
  • Description: Increased IL2 production associated with TLR1/6 presence 10 weeks post BCG vaccination (Randhawa et al., 2011).
  • Detailed Gene Information: Click Here.
• Host IL6 response
  • Description: IL6 expression post vaccination expressed in higher amounts in presence of TLR1/6 (Randhawa et al., 2011).
  • Detailed Gene Information: Click Here.
• Host TLR1 response
  • Description: After stimulation with TLR1 lipopeptide ligands, PBMCs from TLR1-deficient, BCG-vaccinated individuals secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR1 genotypes (Randhawa et al., 2011).
  • Detailed Gene Information: Click Here.

Mouse Response

• Host Strain: C57BL/6, 8-10 wk female
• Vaccination Protocol: Mice were injected subcutaneously in flanks with 100μL or intranasal with 5μL/nostril of antigen plus adjuvant. The control BCG vaccination involved subcutaneous injection of 5x10^5 CFU in 100μL PBS in hind flank. Two formulations of the adjuvant were used, such that mice were either treated at day 0 with AMOs-TT conjugate in Eurocine L3 emulsion or suspension, followed by nasal booster at day 21 (Hamasur et al., 2003).
• Immune Response: Immunization with AMO-TT, AMOs-Ag85B, or AMOs-75kDa conjugate resulted in increased lymphocyte proliferation in spleen when responding to PPD (Hamasur et al., 2003).
• Challenge Protocol: Mice were challenged intranasally with 10^5 M. tuberculosis Harlingen on day 24 (vaccination on day 0).
• Efficacy: All mice started to die after 17 weeks (median time = 36 weeks) (Hamasur et al., 2003). Considerable increases in weight were observed for mice immunized with AMOs-TT conjugate or BCG versus those not vaccinated (Hamasur et al., 2003). Mice immunized with AMOs-TT conjugate and either adjuvant showed longer survival than sham-immunized mice. The emulsion adjuvant showed increased protective efficacy versus the suspension version (Hamasur et al., 2003)

Mouse Response

• Host Strain: C57BL/6, 8-10 wk female
• Vaccination Protocol: Mice were immunized subcutaneously on days 0, 28 with 5 or 25 μg of AMO-Ag85B in 1% alum. Control mice were vaccinated s.c. with live BCG on day 0.
• Side Effects: Reduced weight loss observed for BCG and AMO-Ag85B immunized mice versus shams (Hamasur et al., 2003).
• Challenge Protocol: Mice were challenged intravenously with 10^5 CFU M. tuberculosis H37Rv on day 60(Hamasur et al., 2003).
• Efficacy: BCG-immunized mice exhibited best survival versus mice immunized with the AMO-Ag85B vaccine. However, AMO-Ag85B immunized mice also showed significant improvement in survival versus sham-immunized mice.

Mouse Response

• Host Ifng (Interferon gamma) response
  • Description: Immunization with BCG[pMV361] (BCG with empty pMV361) vaccine induced significant up regulation of IFN-gamma in mice 45 days after immunization as compared to non-immunized mice. Cytokines were measured in lymphocytes from the spleen (Singh et al., 2011).
  • Detailed Gene Information: Click Here.
• Host IL-6 response
  • Description: Immunization with BCG[pMV361] (BCG with empty pMV361) vaccine induced significant up regulation of IL-6 in mice 45 days after immunization as compared to non-immunized mice. Cytokines were measured in lymphocytes from the spleen (Singh et al., 2011).
  • Detailed Gene Information: Click Here.
• Host Il2 response
  • Description: Immunization with BCG[pMV361] (BCG with empty pMV361) vaccine induced significant up regulation of IL-2 in mice 45 days after immunization as compared to non-immunized mice. Cytokines were measured in lymphocytes from the spleen (Singh et al., 2011).
  • Detailed Gene Information: Click Here.
• Host TNF-alpha response
  • Description: Immunization with BCG[pMV361] (BCG with empty pMV361) vaccine induced significant up regulation of TNF-alpha in mice 45 days after immunization as compared to non-immunized mice. Cytokines were measured in lymphocytes from the spleen (Singh et al., 2011).
  • Detailed Gene Information: Click Here.

Mouse Response

• Host Strain: C57BL/6
• Vaccination Protocol: Ten to twelve female C57BL/6 mice were immunized per group with 100 μl of the formulation, once, subcutaneously (s.c.) at the base of the tail. BCG and Lactoferrin were emulsified with Freund's adjuvant in a 1:1 ratio (Hwang et al., 2005).
• Immune Response: A single immunization of mice with Lactoferrin as an adjunct adjuvant resulted in amplified splenocyte proliferative response to heat-killed BCG, and elevated IL-12(p40) production with increased relative ratios of IL-12/IL-10. Furthermore, splenocyte recall response to HK-BCG was augmented for proinflammatory mediators, TNF-alpha, IL-1beta, and IL-6, approaching responses generated to complete Freund's adjuvant (CFA) immunized controls (Hwang et al., 2005).
• Challenge Protocol: Fourteen days post-immunization, four mice from each group were aerosol challenged with Erdman MTB (Hwang et al., 2005).
• Efficacy: All immunization groups showed significant reduction in lung organism load, decreased bacterial load in the spleen (Hwang et al., 2005).
• Host Ifng (Interferon gamma) response
  • Description: Lactoferrin was able to significantly augment the production of IFN-γ compared to vaccine preparations in IFA alone (Hwang et al., 2005).
  • Detailed Gene Information: Click Here.
• Host IL-1b response
  • Description: In concert with the increased stimulation index, there was significant production of all three proinflammatory mediators (TNF-alpha, IL-1beta, and IL-6) in the Lactoferrin immunization group compared to both the non-immunized and IFA immunized groups (Hwang et al., 2005).
  • Detailed Gene Information: Click Here.
• Host IL-6 response
  • Description: In concert with the increased stimulation index, there was significant production of all three proinflammatory mediators (TNF-alpha, IL-1beta, and IL-6) in the Lactoferrin immunization group compared to both the non-immunized and IFA immunized groups (Hwang et al., 2005).
  • Detailed Gene Information: Click Here.
• Host Il12b response
  • Description: A single administration of BCG in IFA and Lactoferrin resulted in significantly increased production of IL-12 in the splenic recall assay (Hwang et al., 2005).
  • Detailed Gene Information: Click Here.
• Host Ltf response
  • Description: Lactoferrin can act as an adjunct adjuvant to augment cellular immunity and boost BCG efficacy for protection against subsequent challenge with virulent MTB. Augmented responses were found for IL-12, TNF-alpha, IL-1beta, and IL-6 (Hwang et al., 2005).
  • Detailed Gene Information: Click Here.
• Host TNF-alpha response
  • Description: In concert with the increased stimulation index, there was significant production of all three proinflammatory mediators (TNF-alpha, IL-1beta, and IL-6) in the Lactoferrin immunization group compared to both the non-immunized and IFA immunized groups (Hwang et al., 2005).
  • Detailed Gene Information: Click Here.

Rat Response

• Host Strain: Wistar rat
• Vaccination Protocol: Wistar rats were vaccinated with 10^6 BCG subcutaneously 6-8 weeks prior to challenge (Singhal et al., 2011).
• Vaccine Immune Response Type: VO_0000286
• Immune Response: A significantly greater number of total cells and CD8 T cells was observed in BCG vaccinated animals at day 60 post-challenge compared with unvaccinated animals. Immunohistochemistry revealed substantial higher number of CB8 cells in the lung lesions of vaccinated rats at day 30. Finally, a high number of mycobacteria-specific CD4 and CD8 T cells producing IFN-gamma was detected in BCG vaccinated rats (Singhal et al., 2011).
• Challenge Protocol: The rats were challenged with a low dose of Mtb W4 strain 6 weeks after vaccination (Singhal et al., 2011).
• Efficacy: A significantly reduced bacillary load was observed in the lungs of BCG vaccinated rats over the entire course of infection. About a 1 log(10) reduced bacillary load was observed in the lungs of vaccinated rats compared to unvaccinated animals (Singhal et al., 2011).
• Description: The study showed that BCG vaccinated Wistar rats efficiently control early bacillary growth and pathology related to Mtb infection (Singhal et al., 2011).

Rabbit Response

• Host Strain: White New Zealand female rabbits (2.5-3kg)
• Vaccination Protocol: Rabbits were immunized with LAM (50 μg), AMO-TT (20 μg), AMO-Ag85B (20 μg), or AMO-75kDA (20 μg) conjugates via i.m. injection with 100 μl PBS and 100 μl FIA emulsifier, and later boosted (weeks 2, 4) with same dose of the previous vaccine.
• Immune Response: LAM alone did not induce LAM IgG antibodies, whereas the three conjugate vaccines induced high IgG antibody titers (Hamasur et al., 2003).
• Challenge Protocol: No challenge protocol implemented for rabbit immunogenicity study.
• Description: Purpose of the experiment with rabbits was to determine immunogenicity response to LAM and AMO-protein conjugates.

Rabbit Response

• Host Strain: New Zealand White Rabbits
• Vaccination Protocol: Rabbits were vaccinated three times at 3-week intervals intramuscularly with a total of 500 micro liters of BCG (Tsenova et al., 2006).
• Persistence: Rabbits were euthanized 8 weeks post-infection (Tsenova et al., 2006).
• Vaccine Immune Response Type: VO_0000134
• Immune Response: Significantly smaller amounts of CFU of the bacterium were observed in the brains of rabbits that were vaccinated with BCG. No granulomas were found in the lungs of mice vaccinated with BCG while they were found in the unvaccinated group. Vaccination with BCG induced a statistically significant increase in antigen-specific T-cell proliferation compared to that of non-vaccinated controls (Tsenova et al., 2006).
• Challenge Protocol: Ten weeks after immunization with BCG the rabbits were anesthetized and immobilized and a spinal needle was used to withdraw .3 mL of cerebrospinal fluid and .2 mL of 5X10^5 CFU of M. tuberculosis was injected intracisternally (Tsenova et al., 2006).
• Efficacy: This study showed that vaccination with BCG induced protection against M. tuberculosis because rabbits that were vaccinated showed increased T-cell proliferation and no granulomas in the lungs (Tsenova et al., 2006).

Guinea pig Response

• Host Strain: Dunkin Hartley, female (250-300g)
• Vaccination Protocol: Guinea pigs were immunized s.c. (day 0) and boosted nasally (day 24) with Eurocine L3 emulsion, AMO-TT conjugate, and variable antigen doses and adjuvant concentrations (Hamasur et al., 2003). A protection study included immunization with 15 μg AMO-Ag85B in 10% Eurocine L3 emulsion. Comparison BCG groups were injected s.c. with 5x10^4 CFU live BCG (Hamasur et al., 2003).
• Side Effects: Five of six animals non-vaccinated and two of six AMO-Ag85B vaccinated were killed before 120 days post-challenge if observed >20% loss of starting weight.
• Challenge Protocol: Guinea pigs were aerosol challenged with saline suspension containing 10^6 organisms per ml (~10 CFU/lung).
• Efficacy: BCG vaccinated animals showed reduced counts of M. tuberculosis bacilli in lung and spleen samples versus saline controls. The reduced CFUs in spleen samples along with histological sections analyzed with double blind analysis suggested an overall decrease in disease severity in lung and spleen (Hamasur et al., 2003).

Guinea pig Response

• Host Strain: Dunkin Hartley (Thom et al., 2012).
• Vaccination Protocol: Pathogen-free, female outbred Dunkin Hartley strain guinea pigs (weighing 500–550 g) were vaccinated subcutaneously with 5 × 104 colony forming units (CFU) of M. bovis BCG Pasteur (Thom et al., 2012).
• Immune Response: Significant down-regulation of both ferritin light- and heavy-chain (Thom et al., 2012).
• Description: PPD from M. tuberculosis was added to splenocytes from BCG-vaccinated guinea pigs at a final concentration of 30 μg/ml for 16 and 24 h at 37 °C in the presence of 5% CO2. The splenocyte cultures from naïve and BCG-vaccinated guinea pigs were infected with M. tuberculosis at an MOI of 0.2 and incubated for 4, 16 and 24 h at 37 °C in the presence of 5% CO2. The viability of uninfected and infected splenocytes from the same vaccination group was determined at 4, 16 and 24 h post-M. tuberculosis infection by trypan blue exclusion (Thom et al., 2012).
• Host Fth1 response
  • Description: Splenocytes from BCG-vaccinated guinea pigs were stimulated ex vivo with purified protein derivative from M. tuberculosis and a significant down-regulation of ferritin light- and heavy-chain was measured by reverse-transcription quantitative-PCR (Thom et al., 2012).
  • Detailed Gene Information: Click Here.
• Host Ftl response
  • Description: Splenocytes from BCG-vaccinated guinea pigs were stimulated ex vivo with purified protein derivative from M. tuberculosis and a significant down-regulation of ferritin light- and heavy-chain was measured by reverse-transcription quantitative-PCR (Thom et al., 2012).
  • Detailed Gene Information: Click Here.

Cattle Response

• Host Strain: Friesian Calves
• Vaccination Protocol: Vaccinated subcutaneously in the neck with 2 mL of 2X10^5 cfu BCG
• Persistence: Cattle were examined 154 to 164 days after they had been challenged.
• Vaccine Immune Response Type: VO_0000408
• Immune Response: The cattle that had been vaccinated with BCG showed significantly less numbers of lesions in the lungs and lymph nodes.
• Challenge Protocol: 54 days after vaccination the calves were challenged intratracheally with 2X10^3 cfu M. bovis strain 83/6235.
• Efficacy: Vaccination with BCG induced considerable protection against a challenge with M. bovis, there were significantly fewer animals with Tuberculosis lesions.
• Description: This study showed that killed M. vaccae did not protect against challenge by M. bovis, however, the vaccination of cattle with BCG by the respiratory route protected them against the development of tuberculous lung lesions.
• Host IFNG response
  • Description: In the vaccinated cattle group, increased IFN-gamma was released from the culture of peripheral blood lymphocytes from cattle after stimulation with bovine purified protein derivative (PPD) (Buddle et al., 1995).
  • Detailed Gene Information: Click Here.
• Host IL10 response
  • Description: In the vaccinated cattle group, increased IL-2 was released from the culture of peripheral blood lymphocytes from cattle after stimulation with bovine purified protein derivative (PPD) (Buddle et al., 1995).
  • Detailed Gene Information: Click Here.

Macaque Response

• Host Strain: cynomolgus monkey (M. fascicularis) and rhesus monkey (M. mulatta)
• Vaccination Protocol: The macaques were vaccinated intradermally with 1–4 X 10^5 cfu bacillus Calmette–Guerin
• Vaccine Immune Response Type: VO_0000859
• Immune Response: Showed specific cell-mediated immune responses after bacillus Calmette–Guerin vaccination as observed by skin test, lymphoproliferation, and IFN-gamma production (Langermans et al., 2001).
• Challenge Protocol: 17 weeks after vaccination the monkeys were challenged with 3,000 cfu M. tuberculosis by intratracheal installation of 3 ml of the bacterial suspension. Between 59 and 65 days after infection the animals were killed, and necropsies were undertaken for pathological examination (Langermans et al., 2001).
• Description: This study showed that cynomolgus monkey (M. fascicularis) is a better model than rhesus monkey (M. mulatta) for M. tuberculosis vaccination and infection studies. From a challenge with 3,000 cfu M. tuberculosis, BCG vaccination protects cynomolgus monkeys with >2-log reduction of the bacterial load and diminished pathology, whereas only a minimal BCG vaccination effect was observed in rhesus monkeys. These two species represent the two extremes of BCG-induced protection that is found in humans (Langermans et al., 2001).

Zebrafish Response

• Host Strain: Wildtype AB or adult rag hu1999 mutant fish
• Vaccination Protocol: Fish were vaccinated with ~8X10^3 CFU BCG intraperitoneally or intramuscularly (Oksanen et al., 2013).
• Vaccine Immune Response Type: VO_0003057
• Challenge Protocol: Fish were anesthetized and intraperitoneally injected 3-4 weeks after immunization with ~20-30 bacteria (Oksanen et al., 2013).
• Efficacy: BCG was unable to prevent infection with mycobacteria, however, it improved fish survival lowering the mortality rate from 84.2% to 45.0% (Oksanen et al., 2013).
• Description: BCG is unable to prevent infection in zebrafish, however, it is able to improve fish survival (Oksanen et al., 2013).