Patent Application: US-201415025976-A

Abstract:
the present invention relates to an immunogenic formulation to be used in mammals against human metapneumovirus , consisting of a lyophilisate of 1 × 10 9 ufc / ml of recombinant bcg that expresses the viral protein p of hmpv . the vaccine is derived from the full or partial product of the p gene of hmpv , capable of being used , stabilised and administered in solution without requiring adjuvants . the preparation can also contain combinations of the aforementioned formulations . the formulation is stabilised by means of lyophilisation in order to be subsequently reconstituted in an adjuvant - free saline solution prior to use .

Description:
the present invention consists of an immunogenic formulation against hmpv comprising a recombinant bcg strain expressing the p - protein from human metapneumovirus . this formulation provides effective protection against infection by hmpv and does not generate inflammatory hyper - responsiveness . the formulation of the invention may be delivered alone or mixed with other vaccines , such as an immunogenic formulation against srv , in a mixed dose that would deliver protection against infection and / or complications associated with hmpv and srv . the immunogenic formulation detailed in this invention can be used to prepare vaccines containing live attenuated recombinant bacteria , from axenic cultures at doses between 1 × 10 4 cfu to 1 × 10 10 cfu per dose , especially dosages of 1 × 10 8 cfu per dose are preferred . the formulation will be presented lyophilized with a reconstituting solution which does not require an adjuvant because the adjuvant is the cell envelope composition of the recombinant bacteria . the lyophilized immunogenic formulation of the invention should be conserved between 4 and 8 ° c ., protected from direct and indirect sunlight , presented in two separate vials , one containing the lyophilized recombinant bacteria and another with the reconstituent saline solution , to be mixed prior its administration . since its introduction in 1921 , the vaccine based on mycobacterium bovis bacillus calmette - guérin ( bcg ) has been used in more than one billion people and is currently used for tuberculosis worldwide . the inventors have found that a way to shift the balance of immune response from a th2 - type pro - inflammatory response , whose protection is not stable in time , towards a th1 - type cell - based response , specifically cd8 + cytotoxic t and cd4 + helper t cells , is the use of adjuvants like the components of mycobacterium cell wall . specifically , expressing the immunogenic protein from hmpv in mycobacterium bovis bacillus calmette - guérin ( bcg ). f and g proteins from hmpv are found in the surface of the viral particle and therefore are accessible to circulating antibodies . therefore , these proteins would be the obvious choice for a vaccine based on producing antibodies to prevent infection with hmpv . notwithstanding the foregoing , in the present invention we have chosen to use unexposed capsid proteins as immunogen , specifically p - protein , together with an adjuvant that promotes a th1 - type cell - based response , such as attenuated mycobacterium bovis bcg bacteria . it has been found that capsid proteins as hmpv m2 . 1 promote the activation of cytotoxic lymphocytes ( ctl ). surprisingly , in the present invention is described the use of the p - protein , a not - exposed protein from hmpv virus , which when used as immunogenic protein expressed by mycobacterium allows to formulate an immunogenic composition , which when used as a vaccine or immunogenic composition provides effective protection without unwanted side effects such as inflammatory hyper - responsiveness . the recombinant proteins expressed by the bacteria vector used herein are obtained by cloning genes encoding the same from group a hmpv virus in prokaryotic expression vectors , all commercially available . metapneumovirus ( hmpv ) genome has been described previously and is available in genbank database , accession number : ab503857 . 1 , dq843659 . 1 , dq843658 . 1 , ef535506 . 1 , gq153651 . 1 , ay297749 . 1 , ay297748 . 1 , af371337 . 2 , fj168779 . 1 , fj168778 . 1 , nc_004148 . 2 and ay525843 . 1 . obtaining the recombinant bacterial strain described herein includes heterologous expression of the viral proteins during bacterial replication in the host . heterologous dna sequences are integrated into the bacterial chromosomal dna through expression vector pmv361 . these hmpv proteins may be genetically engineered , such as by incorporation of peptide sequences or glycosylation domains and / or subsequent destination to endocytic or phagocytic receptors of antigen presenting cells by their coupling to natural ligands using the biotin - streptavidin system ( using commercial biotinylation kits and genetic modification of viral proteins by coupling to bacterial protein streptavidin , or by introducing the genetic sequence encoding for amino acid sequences described as biotinylation signals in recombinant proteins or protein fragments , using techniques previously described for prokaryotic and / or eukaryotic heterologous expression systems ). immunogenic formulation of the invention comprises the expression of hmpv p - protein either complete or an immunogenic fragment thereof , in an attenuated strain of mycobacterium . especially preferred is the recombinant attenuated mycobacterium strain derived from bacillus calmette - guérin ( bcg ) strain . hmpv p - protein can be p - protein from metapneumovirus subtypes a or b . the recombinant attenuated mycobacterium strain of the invention can contain nucleic acid sequences encoding for hmpv p - protein or an immunogenic fragment thereof inserted into the bacterial genome or into an extrachromosomal plasmid , in one or more copies . the protein expression can be under the control of constitutive or inducible , endogenous or exogenous promoters from mycobacterium bcg . the hmpv p - protein or an immunogenic fragment thereof can be expressed in soluble , soluble - cytoplasmic form , as extracellularlly secreted or membrane - bound protein . the immunogenic formulation of the invention may comprise two or more recombinant attenuated strains of mycobacterium , wherein the proteins or immunogenic fragments of hmpv p - protein from the strains are expressed to generate a different number of copies , are constitutively or inducible expressed , and / or are located in different cellular locations . the immunogenic formulation disclosed herein can be used in conjunction with other immunogenic formulations comprising further antigen formulations against different virus of hsrv and hmpv , with attenuated strains of bcg and seasonal vaccines against influenza a and b . the above described immunogenic formulation can be applied to individual in subcutaneous / subdermal form in conjunction with a buffered saline ( pbs , sodium phosphate buffer ) or saline solution . to develop the recombinant mycobacterium bacteria expressing hmpv p - protein , first we must generate a vector expressing said protein . any known mycobacterial expression vector can be used , and the gene encoding for the p - protein from hmpv virus must be inserted in phase with its promoter region . in a preferred embodiment , a reverse transcription with universal primers from the rna from isolated human metapneumovirus is performed . subsequently the p - protein gene is amplified with specific primers ( fig1 a ). the amplification product is inserted into the expression vector . in the present invention we have used the mycobacterial expression vector pmv361 , generating the vector pmv361 - p - hmpv ( fig1 b ). the vector , once obtained , is used to transform a mycobacterium strain , such as mycobacterium bcg . for the purposes of the invention any known bcg strain can be used , such as bcg pasteur or bcg danish . the method of transformation can be any method available , especially preferred is electrotransformation . subsequently , the transformed cells expressing hmpv p - protein must be selected . these recombinant cells are the immunogenic formulation of the invention , which is provided in amounts from between 10 4 to 10 9 cfu / dose in a pharmacologically appropriate saline buffer . in addition , the formulation of the invention can be formulated in combination with recombinant bcg strains expressing immunogenic proteins from respiratory syncytial virus ( rsv ). in a preferred embodiment , the recombinant bcg bacteria expressing the hmpv p - protein is provided in conjunction with recombinant bcg bacteria expressing n , p , m , f , m2 ( orf1 and 2 ), sh , g or l proteins from srv . in a more preferred embodiment , the immunogenic formulation of the invention is used in combination with a bcg strain recombinant for n gene from rsv . particularly , the combination of bcg recombinant for the p gene from hmpv subtype a and bcg recombinant for the n gene of rsv subtype a is preferred . in another embodiment of the invention a bcg strain is transformed with a vector expressing the hmpv p - protein and then the transformed cell is re - transformed with a vector expressing a protein of respiratory syncytial virus ( rsv ). rsv protein is selected from n , p , m , f , m2 ( orf1 and 2 ), sh , g or l . particularly is firstly preferred a transformation with a vector expressing the p - protein from hmpv subtype a , and secondly subjecting these transformed strains to a second transformation with a vector expressing the n gene of rsv subtype a , obtaining a bcg strain simultaneously expressing p - protein from hmpv subtype a and n - protein from srv subtype a . the immunogenic formulation of the invention can be provided lyophilized in a multidose presentation ready for reconstitution in 1 ml of an attached saline solution to obtain 1 × 10 9 cfu / ml of reconstituted solution . each 0 . 1 ml of resuspended solution will contain the appropriate dose of 1 × 10 8 cfu for administration . to prepare the lyophilized solution transformed strains of the invention can be suspended in any appropriate lyophilization solution , for example lyo c buffer comprising 4 % ( w / v ) mannitol , 0 . 05 % ( w / v ) tyloxapol , 0 . 25 % sucrose and 5 mm histidine can be used . then it can be lyophilized and conserved at 25 ° c . the reconstituent solution can be any available in the art , in one embodiment a dilute sauton ssi solution ( 125 μg mgso 4 , 125 μg k 2 hpo 4 , 1 mg l - asparagine , 12 . 5 μg ferric ammonium citrate , 18 . 4 mg 85 % glycerol , 0 . 5 mg citric acid in 1 ml h 2 o ) at − 80 ° c . is preferred . in another embodiment , the formulation of the invention can be suspended in pbs ( 137 mm nacl ; 2 . 7 mm kcl ; 4 . 3 mm na2hpo4 ; 1 . 47 mm kh2po4 , ph 7 . 4 ), supplemented with 20 % glycerol , 0 . 02 % tween 80 at a final concentration of 10 8 bacteria per 100 μl and conserved at − 80 ° c . both in the presence of a nonionic detergent such as 0 . 1 % tween - 80 or 0 . 05 % tyloxapol . the formulation of the invention should be kept between 4 - 8 ° c . before and after reconstitution , kept away from direct and indirect sunlight all the time and upon reconstitution it should be discarded at the end of the day . the following examples for generating and using the immunogenic formulation against hmpv based on the recombinant mycobacterium bacteria expressing viral proteins are illustrative only and are not intended to limit the production or application scope of the invention . although specific terms are used in the following descriptions , its use is only descriptive and not limiting . generation of a recombinant vector allowing the expression of the hmpv p - protein in mycobacterium bcg the coding region of metapneumovirus p gene was amplified from rna using the following primers : p - hmpv_fw : gaattcatgtcattccctgaaggaaa ( seq id no 1 ) and p - hmpv_rv : gaattcctacataattaactggtaaa ( seq id no 2 ), where the underlined sequences incorporate the ecori site at both ends of the amplification product , fig1 a . mycobacterial vector pmv361 was linearized into the ecori site , allowing to insert the sequence corresponding to the p gene from hmpv : the insertion of the open - frame of hmpv p gene into the ecori site of the vector pmv361 locates it under control of the hsp60 promoter and led to the construction of pmv361 - p - hmpv ( fig1 b ). immunogenic formulation comprising 10 doses of 1 × 10 8 cfu each of bcg danish strain recombinant for the p gene from hmpv subtype a the p gene from hmpv subtype a is inserted in one copy into the bacterial genome under regulation of endogenous constitutive hsp 60 promoter from mycobacterium bcg for protein expression . the mycobacterium bcg danish strain ( atcc 35733 ) was transformed by electrotransformation with the plasmid pmv361 - p - hmpv , derived from plasmid pmv361 ( stover et al ., 1991 ), which is inserted once in the bacterial genome . this plasmid contains the gene encoding for the p - protein from hmpv subtype a , which is expressed under the endogenous constitutive promoter of gene hsp60 from bcg . the resulting recombinant colonies were grown ( at 37 ° c . in supplemented middlebrook 7h9 culture medium ( 4 . 9 g / l )) up to od 600 nm = 1 , centrifuged at 4000 rpm for 20 min ( eppendorf rotor model 5702 / r a - 4 - 38 ) and resuspended in pbs ( 137 mm nacl , 2 . 7 mm kcl , 4 . 3 mm na 2 hpo 4 , 1 . 47 mm kh 2 po 4 , ph 7 . 4 ) supplemented with 20 % glycerol and 0 . 02 % tween 80 up to a final concentration of 10 8 bacteria per 100 μl and conserved at − 80 ° c . similarly , the strains can be resuspended in a volume solution : 25 % lactose volume and proskauer and beck medium supplemented with glucose and tween 80 ( pbgt : 0 . 5 g asparagine , 5 . 0 g monopotassium phosphate , 1 . 5 g citrate magnesium , 0 . 5 g potassium sulfate , 0 . 5 ml tween 80 and 10 . 0 g glucose per liter of distilled water ) to be lyophilized and then conserved at 25 ° c . using western blot and antibodies for the hmpv p - protein , it can be seen that this strain of bcg constitutively expressed p - protein from hmpv subtype a in the cytoplasm ( fig2 a ). immunization of balb / c mice with a subdermal dose of 1 × 10 6 colony forming units ( cfu ) of rbcg - p - hmpv , as a vaccine , confers protection to these animals against intranasal infection with 10 6 plaque forming units of hmpv subtype a ( fig3 ). the effect of the vaccine of the invention , rbcg - p - hmpv , was compared with a rbcg vaccine expressing another protein from hmpv , m2 protein , and with untransformed bcg ; an unimmunized group was used as control , all groups were intranasally infected with hmpv , an unimmunized , uninfected control was also included . the variation in weight of animals after infection was analyzed , the results are shown in fig3 a . both recombinant bcg vaccines for p ( ) and m2 (♦) proteins from hmpv were protective of weight loss subsequent to animal infection , obtaining a weight similar to uninfected animal ( ). moreover , unimmunized animals ( ) or animals immunized with wild - type bcg ( wt - bcg ) (▪), live - hmpv ( ) or uv - hmpv ( ) suffered loss of body weight post - infection . immune cell infiltration in the airways of animals after infection was also analyzed , which is an indicative parameter of disease development . this was analyzed by the number of infiltrated cells into the airway ( fig3 b ) and through infiltrating cells that being positive for cd11b and gr1 correspond to granulocytes ( fig3 c ). fig3 b shows that only the vaccine of the invention rbcg - p - hmpv prevented lymphocyte infiltration in airways , resulting in an infiltration similar to uninfected animals . moreover , animals unimmunized or immunized with wild - type bcg ( wt - bcg ) or rbcg - m2 - hmpv vaccines suffered infiltration of immune cells in airways post - infection . vaccination with rbcg - m2 conferred incomplete protection in animal airways for immune cells infiltration . additionally , a number of copies of the n - hmpv gene rna in lung cells of different groups under study was analyzed . a greater number of copies of the hmpv virus rna is indicative of greater infection , the results are normalized per β - actin copies . the results are shown in fig3 d . it is appreciated that the number of hmpv virus present in the lungs of unimmunized infected animals is similar to that of infected animals immunized with wt - bcg , live - hmpv or uv - hmpv , i . e ., these immunizations would not confer protection . it is observed that immunization with rbcg - m2 conferred an incomplete protection . in addition , practically there is no virus in infected animals immunized with the vaccine of the invention rbcg - p , with results similar to the group of uninfected animals . this proves that the formulation of the invention effectively protects against infection with hmpv . immunogenic formulation comprising 5 × 10 7 bacteria from mycobacterium bcg danish strain recombinant for the p gene from hmpv subtype a and 5 × 10 7 bacteria from mycobacterium bcg danish strain recombinant for the n gene from rsv subtype a in each bacteria composing the immunogenic formulation , the genes from hmpv and rsv are inserted into the bacterial genome in one copy under regulation of the endogenous constitutive hsp60 promoter from bcg and protein expression is cytoplasmic . the immunogenic formulation is preserved in pbs ( 137 mm nacl , 2 . 7 mm kcl , 4 . 3 mm na 2 hpo 4 , 1 . 47 mm kh 2 po 4 , ph 7 . 4 ) supplemented with 20 % glycerol and 0 . 02 % tween 80 at a final concentration of 10 8 bacteria per 100 μl and conserved at − 20 ° c . bcg danish strains ( atcc 35733 ) were transformed by electrotransformation with plasmid pmv361 - p from hmpv to give the strain of the invention rbcg - p - mpv , and additionally a second group was transformed with pmv361 - n from srv to obtain the transformed strain rbcg - n srv , these plasmids are derived from plasmid pmv361 ( stover et al ., 1991 ), which are inserted into the bacterial genome once . the resulting recombinant colonies were grown at 37 ° c . in a supplemented middlebrook 7h9 culture medium up to od 600 nm = 1 , centrifuged at 4000 rpm for 20 min ( eppendorf rotor model 5702 / r a - 4 - 38 ) and resuspended in pbs ( 137 mm nacl , 2 . 7 mm kcl , 4 . 3 mm na 2 hpo 4 , 1 . 47 mm kh 2 po 4 , ph 7 . 4 ) supplemented with 20 % glycerol and 0 . 02 % tween 80 up to a final concentration of 10 7 bacteria per 100 μl and conserved at − 20 ° c . strains were resuspended in lyo c buffer , comprising 4 % ( w / v ) mannitol , 0 . 05 % ( w / v ) tyloxapol , 0 . 25 % sucrose and 5 mm histidine , then lyophilized and conserved at 4 ° c . using western blot analysis and specific antibodies for the hmpv p - protein or rsv n - protein , it was observed that bcg danish strains recombinantly expressed , p - protein from hmpv or n - protein from srv , subtype a , respectively ( fig2 ). an immunogenic formulation comprising both transformed strains simultaneously conferred immunity against hmpv and rsv virus . immunogenic formulation of 10 4 bacteria from bcg danish strain recombinant for the p gene from hmpv subtype a a bcg danish strain was transformed with the p gene from hmpv subtype a , so that the gene was inserted into the bacterial genome in one copy under the regulation of the endogenous inducible acr promoter from bcg , which is active in response to nitric oxide , low oxygen concentrations , and stationary growth phases . protein expression is cytoplasmic . the immunogenic formulation was lyophilized in lyo c buffer , comprising 4 % ( w / v ) mannitol 0 . 05 % ( w / v ) tyloxapol , 0 . 25 % sucrose and 5 mm histidine and was conserved reconstituted at 25 ° c . in dilute sauton ssi solution ( 125 μg mgso 4 , 125 μg k 2 hpo 4 , 1 mg l - asparagine , 12 . 5 μg ferric ammonium citrate , 18 . 4 mg 85 % glycerol , 0 . 5 mg citric acid in 1 ml h 2 o ). alternatively , the transformed strains can be preserved in pbs ( 137 mm nacl ; 2 . 7 mm kcl ; 4 . 3 mm na 2 hpo 4 ; 1 . 47 mm kh 2 po 4 , ph 7 . 4 ) supplemented with 0 . 02 % tween 80 y 20 % glycerol up to a final concentration of 10 4 bacteria per 100 μl . bcg danish strain ( american type culture collection , www . atcc . orq , atcc number 35733 ) was transformed by electrotransformation with the plasmid pmv361 pacr / hmpv derived from the plasmid pmv361 ( stover et al ., 1991 ), which is inserted into the bacterial genome once . this plasmid contains the gene encoding for the p - protein from hmpv subtype a , which is expressed under the endogenous inducible acr promoter from bcg . the resulting recombinant colonies were grown ( at 37 ° c . supplemented middlebrook 7h9 culture medium ) up to od 600 nm = 1 , centrifuged at 4000 rpm for 20 min ( eppendorf rotor model 5702 / r a - 4 - 38 ) and resuspended in a lyo c buffer solution , composed of 4 % ( w / v ) mannitol , 0 . 05 % ( w / v ) tyloxapol , 0 . 25 % sucrose and 5 mm histidine . finally , 1 ml aliquots with 10 4 bacteria were lyophilized and conserved at 25 ° c . similarly , the strains can be preserved in pbs ( 137 mm nacl ; 2 . 7 mm kcl ; 4 . 3 mm na 2 hpo 4 ; 1 . 47 mm kh 2 po 4 , ph 7 . 4 ) supplemented with 0 . 02 % tween 80 and 20 % glycerol at a final concentration of 10 4 bacteria per 100 μl . this immunogenic formulation confers immunity against hmpv virus . immunogenic formulation consisting of 10 9 bacteria from bcg danish strain recombinant for the gene p from hmpv subtype a the gene is inserted into the bacterial genome in one copy , under regulation of the exogenous t7 - phage promoter for constitutive expression in bcg strains that co - express t7 - phage polymerase . protein expression is cytoplasmic . the immunogenic formulation is suspended in dilute sauton ssi solution ( 125 μg mgso 4 , 125 μg k 2 hpo 4 , 1 mg l - asparagine , 12 . 5 μg ferric ammonia citrate , 18 . 4 mg 85 % glycerol , 0 . 5 mg citric acid in 1 ml h 2 o ) and was conserved at 20 ° c ., or can be lyophilized and conserved at 4 ° c . similarly , the strains can be preserved in pbs ( 137 mm nacl ; 2 . 7 mm kcl ; 4 . 3 mm na 2 hpo 4 ; 1 . 47 mm kh 2 po 4 , ph 7 . 4 ) supplemented with 0 . 02 % tween 80 and 20 % glycerol up to a final concentration of 10 9 bacteria per 100 μl . the bcg danish strain atcc 35733 was transformed by electrotransformation with the plasmid pmv361 pt7 / p - hmpv derived from the plasmid pmv361 ( stover et al ., 1991 ), which is inserted into the bacterial genome once . this plasmid contains the gene encoding the p - protein from hmpv subtype a , which is expressed under t7 promoter activated by the expression of the t7 - phage polymerase . the resulting bcg strain was transformed by electrotransformation with the plasmid pmv261 amp / polt7 derived from the plasmid pmv261 ( stover et al ., 1991 ), which resides extrachromosomally in multiple copies inside the bacteria . in this plasmid , the resistance against the antibiotic kanamycin has been replaced by resistance against antibiotic hygromycin ( hygr ). the t7 polymerase from the t7 phage is under control of the constitutive promoter of the hsp60 gene from bcg . the resulting recombinant colonies were grown at 37 ° c . in supplemented middlebrook 7h9 culture medium up to od 600 nm = 1 , centrifuged at 4000 rpm for 20 min ( eppendorf rotor model 5702 / r a - 4 - 38 ) and resuspended in dilute sauton ssi solution ( 125 μg mgso 4 , 125 μg k 2 hpo 4 , 1 mg l - asparagine , 12 . 5 μg ferric ammonium citrate , 18 . 4 mg 85 % glycerol , 0 . 5 mg citric acid in 1 ml h 2 o )) and conserved at − 80 ° c . this immunogenic formulation confers immunity against hmpv virus . inoculation of 10 8 bacteria from bcg danish strain recombinant for hmpv - p gene in any of the immunogenic formulation thereof protects from immunopathological damage inoculation of the recombinant bcg strain expressing the p - protein from metapneumovirus in any of its formulations protects against clinical signs and symptoms of infection with strain a and b of hmpv . decreases anorexia caused by fever and malaise ( fig3 a ), as well as the infiltration of the airways ( fig3 b 3 c ). it also decreases the replication and spread of strains a and b of hmpv ( fig3 d ). inoculation of the formulation in any presentation also significantly reduces histopathological manifestations of infection by strains a and b of hmpv . the lungs of animals immunized with the formulation show a much less infiltration of immune cells , loss of structure and inflammation of lung tissue with respect to unimmunized animals or animals immunized with the wild - type strain ( fig4 ). inoculation of the formulation with the recombinant bcg expressing the hmpv p - protein in any presentation generates a cell - based immunity that can be transferred to naïve individuals by recovery and purification of t lymphocytes from immunized donors ( fig6 ). the above examples are extended to immune formulations containing a recombinant attenuated mycobacterium strain expressing hmpv p - protein or a substantial part of the same , as well as all combinations with immunological formulations containing a recombinant attenuated mycobacterium strain expressing any of ns2 , n , p , m , sh , m2 ( orf1 ), m2 ( orf2 ), l , f or g proteins from rsv . also examples are extended to immunological formulations which contain one or several recombinant attenuated mycobacterium strains ; wherein said recombinant bacteria contains protein genes , or immunogenic fragments of the hmpv p - protein embedded in either the bacterial genome or extrachromosomal plasmids , in one or more copies , and the expression thereof is commanded by endogenous or exogenous , constitutive or inducible promoters , and it is expressed in a cytoplasmic - 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