there has been a striking upsurge in tuberculosis ( tb ) vaccine research in china in recent years .
more than 80 papers reporting such research have been published since 2004 ( figure 1 ) , many in english language journals .
this has been driven and funded largely by chinese government agencies , since the scale of the tb problem and the difficulties in implementing fully effective control measures were recognized .
, we can note that there are at least two additional restricting factors limiting the progress of the field in china : few research groups have access to facilities for safely working with animals infected with a dangerous pathogenic organism such as the tubercle bacillus ; infrastructure for undertaking vaccine clinical trials to international standards is sparse . in consequence ,
the universal problem of how to select among the promising approaches and candidate vaccines is particularly acute in china .
nevertheless , the basic research problems are being tackled with some enthusiasm and creativity , and the new 5-year program to improve tb control that was announced by the chinese ministry of health on 1 april 2009 will have an impact .
the antigens investigated to date and the locations of research groups are listed in tables 1 and 2 , respectively .
a substantial body of work has gone into characterizing the immune responses to be found in tb infection and following vaccination .
the twin purposes of identifying candidate antigens for use in vaccines and diagnostic tests and of defining immunological markers for latent infection and disease states drive these studies .
these studies have not always been followed up with tests of protective or therapeutic efficacy in animal infection models .
the increased immunogenicity of mouse dendritic cells transfected with antigen heat shock protein 65 ( hsp65 ) has been tested in wuhan and in hong kong , and differences have been found in the polarizing effect of intracellular mycobacterium bovis bacille calmette  guerin ( bcg ) vaccine bacteria on antigen presentation by human adult and cord blood dendritic cells .
various forms of recombinant bcg expressing early secreted antigen 6 of mycobacterium 
 tuberculosis ( esat6 ) , or antigen 85b ( ag85b ) and antigen rv3425 , or ag85b and esat6 with tumor - necrosis factor- , or esat6 and human granulocyte macrophage - colony stimulating factor ( gm - csf ) have shown enhanced immunogenicity .
recombinant m. smegmatis expressing m. tuberculosis culture filtrate protein 10 ( cfp10)/esat6 fusion protein was found stimulatory for macrophage inducible nitric oxide synthetase .
recombinant m. smegmatis and bcg strains have been made that can express cloned antigens at a range of different levels under the control of modified fura gene promoters .
other bcg recombinants expressing esat6 , esat6/interleukin 2 ( il-2 ) or ag85b / esat6 fusions have been made .
a range of known antigens has been tested for immunogenicity as mixtures , fusion proteins and peptides .
these include chimeric ag85b / esat6 with adjuvants monophosphoryl lipid a ( mpl ) and trehalose 6,6'-dimycolate , hsp16.3 with dimethyl - dioctadecyl - ammonium bromide / mpl ( dda / mpl ) adjuvant , fusion protein m. tuberculosis protein 64 ( mpt64)esat6 , resuscitation - promoting factor b ( rv1009 ) , or cfp10 , esat6 or rpfe ( rv2450 ) with nitrocellulose , or rv3772 and rv3425 with incomplete freund 's adjuvant ; or ag85b / mpt64190198/mtb8.4 plus a novel adjuvant of dda with bcg extract .
in - silico analysis of putative mhc class 1-restricted epitopes present in antigens encoded within the region of difference 1 ( rd-1 ) to rd-16 regions of m. bovis genome has revealed potential high - affinity hla binders and profiles of human humoral responses to 38-kda , mtb48 , cfp10/esat6 antigens have been defined .
screening of human immune sera against an expression library of m. tuberculosis open reading frames revealed three novel antigens among the top 20 most strongly recognized : rv1987 , rv3807c and rv3887c .
more than a dozen studies have used dna vaccination as a means of delivering antigens in immunogenicity tests . many have shown th1-biased immunogenicity without additional adjuvanting , for example with ag85b , ag85b / esat6 fusion , esat6/cfp10 fusion , mtb8.4/38-kda / ag85b fusion and epitopes from esat6 , ag85a , cfp10 and ag85b inserted within hsp65 .
targeting the expressed product for degradation via the ubiquitin pathway has been used to enhance mhc class 1 presentation of epitopes from mpt64 and 38-kda , mpt64 , and esat6 .
enhanced responses to encoded antigens have been obtained by additionally encoding cytokines , such as interleukin 21 ( il-21 ) , with ag85a or ag85a / esat6 , and gm - csf with ag85a .
inclusion of dna expressing il-12 enhanced prime / boost responses to bcg and to plasmids expressing ag85a and esat6 .
esat6 dna priming and protein boosting has also been shown to give enhanced th1 responses .
many antigen preparations have been tested for their capacity to protect against challenge infection with virulent m. tuberculosis .
subcutaneous esat6/cfp10 , or esat6/mpt64 fusion proteins on nitrocellulose protected mice against h37rv challenge , but not as effectively as bcg .
hsp65/il-2 fusion protein was found to elicit better protection than hsp65 given with dda / mpl adjuvant and protection was equivalent to that produced by bcg .
similarly , either hspx , a dormancy associated antigen , or synthetic epitope 91104 gave protection equivalent to bcg when given with dda / mpl .
/ mpt64190198/mtb8.4 fusion protein with dda adjuvant boosted protection against challenge in bcg primed mice .
a similar fusion protein in which mtb8.4 was replaced by hspx gave a similar boost , but boosting with a mixture of the two fusion proteins was even better .
a fusion protein of esat6 and ag85a also significantly boosted protection against h37rv in mice .
recombinant bcg expressing the ag85b / mpt64190198/mtb8.4 fusion protein gave slightly better protection to mice against h37rv challenge than parent bcg or bcg expressing rag85b alone .
recombinant bcg expressing a fusion protein of human interleukin ( hil)-12p70 and esat6 showed increased immunogenicity but less protective effect .
recombinant salmonella typhimurium that both expressed esat6/ag85b fusion protein and delivered it as a dna vaccine when given orogastrically gave protection similar to subcutaneous bcg and the combination was superior to either vaccine alone .
the earliest reports indicated protection in mice superior to bcg when a divalent construct expressing both ag85b and mpt64 , or a mixture of plasmids expressing ag85b , mpt64 , mpt63 and esat6 was used . the protection given by a mixture of three plasmids expressing mpt83 , ag85b and esat6
was enhanced by including dda adjuvant and an encoded fusion protein of ag85b and mpt64 was superior to the separately encoded antigens .
encapsulation in poly(lactide - co - glycolide ) microspheres with dda enhanced the protective efficacy in mice of dna - encoding ag85b / mpt64/mpt83 fusion antigen , and strikingly the dna mixed with dda was superior to bcg in protecting cattle against challenge .
inclusion of a plasmid expressing il-2 improved protection by this plasmid or by plasmid expressing mtb8.4 .
a mixture of plasmids encoding ag85b , mpt64 , mpt70 and tb10.4 boosted protection by bcg , as did a plasmid expressing a cfp21/mpt64 fusion protein .
ag85b or ag85a were superior to esat6 when compared separately for protection as dna vaccines .
dna expressing a fusion of mpb64/ag85b / esat6 was superior to a mixture of plasmids expressing the separate antigens and gave protection equivalent to bcg .
the protective effect of dna expressing hsp65 against bcg challenge was enhanced by incorporating epitopes of esat6 , ag85a / b and cfp10 within the hsp65 backbone .
the protective effect of hsp65 dna against h37rv challenge was increased by expression as a fusion with hil-2 , but did not surpass that of bcg .
expression of ag85b fused to bovine herpes virus 1 vp22 protein , which facilitates dissemination of antigen to adjacent cells , resulted in protection against h37rv challenge in mice that was better than protection by bcg .
few studies have been conducted with animals other than mice : a mixture of ag85b , hspx and cfp10/esat6 fusion together with cpg and aloh as adjuvant was immunogenic in mice but gave little protection against challenge with h37rv in guinea pigs ; in contrast , combined dna vaccines encoding antigens ag85b , mpt64 and mpt83 given with dda appeared to be better than bcg in protecting cattle .
interest in therapeutic vaccination has been sustained by clinical studies of a commercial chinese product , m. vaccae extract .
recent meta - analyses of published data concluded that this product gave significant benefit in preventing tb in people at high risk ( 13 studies ) , but there was only a minor benefit from treating new tb cases ( 54 studies ) . in a unique and contrasting approach
, a recombinant m. smegmatis delivering dna expressing human granulysin and murine il-12 was recently found to be therapeutic against h37rv infection .
most research into therapeutic vaccines for tb has focused on the use of naked dna vaccination .
dna had a significant therapeutic effect against h37rv in mice and the fusion hsp65/il-2 was significantly better .
treatment of infected mice with a dna vaccine expressing a fusion protein of mycobacterial hsp70 and leukocyte cluster of differentiation antigen 80 substantially reduced acid - fast bacteria and pathology in liver and spleen , whereas bcg had no effect .
although treatment with dna expressing ag85b was therapeutic , treatment with dna expressing mpt64 was not , and the mixture was less effective than the ag85b vaccine on its own , and inclusion of dna expressing il-12 gave a slight additional benefit .
mice infected with a clinical isolate resistant to isoniazid and rifampicin responded well to treatment with the drugs plus dna expressing chimeric ag85a / esat6 , and appeared to respond better to treatment with the drugs plus dna expressing ag85a than to the dna vaccine alone ; ag85a dna alone was at least as effective as ag85a plus rifampicin in treating mice infected with a strain resistant to rifampicin and isoniazid , ag85a
an immunogenic mixture of dna vaccines expressing ag85b , mpt64 and mpt83 has been found to work as a potent adjunct to isoniazid plus pyrazinamide therapy in mice and to be therapeutic in cattle infected with m. bovis , reducing both pathology and bacterial numbers ; a mixture expressing ag85b , mpt64 and hsp65 was similarly effective .
inclusion of immunostimulatory nucleotide motifs in the gene transcript enhanced the therapeutic efficacy of a plasmid expressing hsp65 when tested against h37rv in mice .
it is evident in considering this body of recent tb vaccine research in china that much of it has been tactical in nature , establishing credentials both nationally and internationally , and building new research bases . additionally , there are creative and insightful studies of particular relevance to the needs of china .
researchers are now able to exploit cutting edge technologies in designing new tb vaccines and are increasingly able to test the vaccines in relevant animal models of infection .
the evidence that dna vaccines can provide effective therapy for tb in cattle may be a significant pointer to the future .
the potential benefits of adding immunotherapies / therapeutic vaccines to tb chemotherapy have been recognized in china , but both preventive and therapeutic approaches against human tb await development of clinical trial capacity for their proper assessment .
 it is now privately acknowledged that there may be little if any perceptible impact of the national bacille calmette  guerin ( bcg ) vaccination program on disease prevalence , despite the extensive coverage of the newborn infant population and likely benefit in the early years of life . a better preventive vaccine than bcg is now being sought by chinese researchers . 
 urgency has been added to the control problem by the emergence of multidrug - resistant tuberculosis ( tb ) . 
 furthermore , expensive second - line drugs seem unlikely to be made available by the government to treat drug - resistant cases , so attention in addition has turned to the potential of immunotherapy as an adjunct to chemotherapy . 
 research trends are summarized here . 