each year , streptococcus pneumoniae causes more than 800,000 deaths worldwide in children under 5  years of age .
currently marketed s. pneumoniae vaccines , which are based on polysaccharide capsular antigens from the most common strains , have substantially reduced pneumococcal disease rates . however , because serotypes can vary between countries or regions , coverage may be incomplete in some cases . moreover
, serotype replacement might eventually render these vaccines less effective . to provide broader , more diverse , and possibly infection stage - specific protection , vaccines based on conserved proteins
are being investigated . , pneumococcal histidine triad protein d ( phtd ) is a conserved surface protein that mediates attachment to respiratory epithelial cells and can elicit a protective immune response . in mice ,
intranasal immunization with phtd generates robust serum antibody and cd4 th1-biased immune memory responses and confers protection against pneumococcal colonization .
a second study in mice showed that vaccination with phtd protects against nasopharyngeal and lung colonization . in a primate study , vaccination with phtd and chemically detoxified pneumolysin induced high levels of antibodies and protected against a challenge with s. pneumoniae serotype 19f .
a phase i trial in adults 1850  years of age showed that a aluminum phosphate - adjuvanted phtd vaccine candidate was well tolerated , immunogenic , and could be boosted by a second vaccine dose . during development of an enzyme - linked immunosorbent assay ( elisa ) to measure antibody responses in the phase i trial , we found that individual and pooled serum from unimmunized healthy adults contained substantial phtd - binding antibody ( data not shown ) . to further investigate the immune response elicited by a phtd - based pneumococcal vaccine
, we developed a murine passive protection sepsis model for assessing the functional activity of human anti - phtd antibodies . naturally occurring human phtd - binding antibody
was purified from a commercial pooled serum ( obtained from approximately 200 healthy individuals ; sigma , st .
the concentration of anti - phtd antibody was determined by elisa , and its purity and specificity were confirmed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis , western blotting , and competition with recombinant phtd ( data not shown ) .
the purified phtd - binding antibodies were passively transferred by intraperitoneal injection ( 200  l ) to 6- to 8-week - old female cba / cahn - btk xid /j
( cba / n ) mice ( jackson laboratories , bar harbor , me ) .
after 1  h , the mice were challenged by intravenous injection with a lethal dose ( 50 colony forming units [ cfu ] in 200  l ) of s. pneumoniae strain a66.1 ( serotype 3 ) ( obtained from d. briles , university of alabama - birmingham ) . the proportion of mice surviving at 14  days post - challenge increased with the concentration of anti - phtd antibody ( fig . 
the dose providing 50% survival ( ed50 ) was estimated to be 1679 elisa units ( eu)/ml ( 95% confidence interval [ ci ] , 14201946 ) by logistic regression with probit link . 

6- to 8-week - old female nave cba / n mice ( n = 5/group ) received an intraperitoneal injection of 200  l test sample or pbs .
after 1  h , mice were challenged intravenously with a lethal dose of s. pneumoniae strain a66.1 ( serotype 3 ) , and survival was monitored for 14  days .
data were analyzed using logistic regression with probit link under proc glimmix in sas version 8.2 to determine the ed50 . in each plot
, circles indicate survival data for individual samples , and the best fit regression is shown as a green line , with the upper and lower limits of the 95% confidence interval shown as yellow and red lines , respectively .
the horizontal line indicates a median response of 50% survival . in ( a ) ,
mice were injected with 2.827.5  eu ( 110  g ) of purified anti - phtd antibody or pbs .
survival data were from 4 passive protection experiments , which included 13 survival points . in five of the 13 cases ( 206 , 275 , 687 , 1375 , 2063 , and 2750  eu / ml ) ,
the passive protection experiment was performed twice , and for 2 of these ( 275 and 2750  eu / ml ) , the points overlap and appear as a single data point .
protection experiments for 3 cases ( 43 , 435 , and 2178  eu / ml ) were not repeated and were included to provide a more appropriate range of antibody concentrations . in ( b ) , mice were injected with 1:20 to 1:60 post - immune sera . to account for the baseline levels of protection seen in the pre - immune sera , the analysis was corrected for over - dispersion using proc genmod with the scale = option in sas version 9.13 and based on survival data from mice that received pre- and post - immune sera . 
 
6- to 8-week - old female nave cba / n mice ( n = 5/group ) received an intraperitoneal injection of 200  l test sample or pbs .
after 1  h , mice were challenged intravenously with a lethal dose of s. pneumoniae strain a66.1 ( serotype 3 ) , and survival was monitored for 14  days .
data were analyzed using logistic regression with probit link under proc glimmix in sas version 8.2 to determine the ed50 . in each plot , circles indicate survival data for individual samples , and the best fit regression is shown as a green line , with the upper and lower limits of the 95% confidence interval shown as yellow and red lines , respectively .
the horizontal line indicates a median response of 50% survival . in ( a ) ,
mice were injected with 2.827.5  eu ( 110  g ) of purified anti - phtd antibody or pbs .
survival data were from 4 passive protection experiments , which included 13 survival points . in five of the 13 cases ( 206 , 275 , 687 , 1375 , 2063 , and 2750  eu / ml )
, the passive protection experiment was performed twice , and for 2 of these ( 275 and 2750  eu / ml ) , the points overlap and appear as a single data point .
protection experiments for 3 cases ( 43 , 435 , and 2178  eu / ml ) were not repeated and were included to provide a more appropriate range of antibody concentrations . in ( b ) , mice were injected with 1:20 to 1:60 post - immune sera . to account for the baseline levels of protection seen in the pre - immune sera ,
the analysis was corrected for over - dispersion using proc genmod with the scale = option in sas version 9.13 and based on survival data from mice that received pre- and post - immune sera .
we next examined the activity of vaccine - induced antibodies in the passive protection model . in the phase i clinical trial ,
adults were vaccinated on weeks 0 and 4 with 6 , 25 , or 100  g of the candidate aluminum phosphate - adjuvanted phtd vaccine .
as described above , we found substantial pre - existing anti - phtd antibodies in pooled sera from vaccine - nave healthy adults .
we therefore selected subjects who had the lowest pre - immune passive protection activity and the highest post - immune phtd antibody titers for testing in the passive protection assay .
for testing in subsequent experiments , we selected a dilution for each subject where the pre - immune serum was not protective in the passive protection assay .
all samples were initially tested at a dilution of 1:20 , and survival was compared to control mice injected with pbs .
human pre - immune sera that were protective at a 1:20 dilution and for which corresponding post - immune sera ( week 8) had an increase in titer of at least 1000  eu / ml were further tested at a 1:40 dilution .
pre - immune sera shown to be protective at a 1:40 dilution and for which corresponding post - immune sera had an increase in titer of at least 2000  eu / ml over baseline were further tested at a 1:60 dilution . of the 54 serum pairs tested , we identified appropriate dilutions for 18 ( table 1 ) .
vaccination increased the anti - phtd antibody titer in all subjects at all vaccine doses ( 6 , 25 , and 100  g ) .
21 , 36 , 37 , 41 , 43 , 45 , 51 , 57 , 58 , and 61 ) significantly delayed death in the murine challenge model compared to the same dilution of the corresponding pre - immune serum . in addition , 2 of these post - immune sera ( from subject nos .
37 and 43 ) significantly improved survival compared to the corresponding pre - immune serum .
as with the naturally acquired antibody , survival increased with the concentration of anti - phtd antibody as determined by elisa ( fig . 
the ed50 in this case was estimated to be 1331  eu / ml ( 95% ci , 7622038 ) . 

table 1.anti-phtd titers and survival mediated by the 18 selected paired pre-/post - immune seraanti - phtd titer ( eu / ml)mice surviving at day 14 ( n / n)significant difference ( p<0.05)subjectvaccine dosepre - immune serum ( week 0)post - immune serum ( week 8)differenceserum dilution testedpre - immune serum ( week 0)post - immune serum ( week 8)delay to deathprotection46  g163125210881:201/53/566  g853452611:200/50/586  g138225861:200/50/5156  g872141271:200/50/5206  g663632971:201/52/5216  g77157801:200/52/5x236  g2106104001:200/51/52525  g137134912121:401/50/53625  g109136412561:401/54/5x3725  g14510398931:200/55/5xx4125  g204634431:200/50/5x4325  g89247823891:401/55/5xx45100  g69141213431:200/52/5x51100  g256140411481:400/50/5x56100  g302222319211:400/52/557100  g107323931321:600/53/5x58100  g326474344171:406/1010/10x61100  g288323129441:400/104/10xin the phase i clinical trial , healthy adults were vaccinated twice ( week 0 , 4 ) with the candidate aluminum phosphate - adjuvanted phtd vaccine . dilutions ( 1:20 ) of pre - immune sera ( 200  l )
were passively transferred by intraperitoneal injection to 6- to 8-week - old female nave cba / n mice ( n = 5/group ) .
after 1  h , the mice were challenged by intravenous injection of a lethal dose ( 50  cfu in 200  l ) of s. pneumoniae strain a66.1 ( serotype 3 ) .
pre - immune sera that were protective at a 1:20 dilution and for which corresponding post - immune sera ( week 8) had an increase in titer of at least 1000  eu / ml was further tested at a 1:40 dilution .
pre - immune sera shown to be protective at a 1:40 dilution and for which corresponding post - immune sera had an increase in titer of at least 2000  eu / ml over baseline were further tested at a 1:60 dilution . using this method , appropriate dilutions for testing
paired pre - immune and post - immune sera from these subjects were tested in the passive protection assay , and survival after 14  days is shown .
delay to death was assessed by survival distribution functions using the product - limit approach and compared between groups of mice by log - rank test using proc lifetest in sas version 9.13 .
differences in protection were compared by one - sided fisher 's exact test using proc freq with the exact option in sas version 9.13 . 
 
anti - phtd titers and survival mediated by the 18 selected paired pre-/post - immune sera in the phase i clinical trial , healthy adults were vaccinated twice ( week 0 , 4 ) with the candidate aluminum phosphate - adjuvanted phtd vaccine .
dilutions ( 1:20 ) of pre - immune sera ( 200  l ) were passively transferred by intraperitoneal injection to 6- to 8-week - old female nave cba / n mice ( n = 5/group ) .
after 1  h , the mice were challenged by intravenous injection of a lethal dose ( 50  cfu in 200  l ) of s. pneumoniae strain a66.1 ( serotype 3 ) .
survival was followed for 14  days . pre - immune sera that were protective at a 1:20 dilution and for which corresponding post - immune sera ( week 8) had an increase in titer of at least 1000  eu / ml was further tested at a 1:40 dilution .
pre - immune sera shown to be protective at a 1:40 dilution and for which corresponding post - immune sera had an increase in titer of at least 2000  eu / ml over baseline were further tested at a 1:60 dilution . using this method , appropriate dilutions for testing were identified for the 18 subjects shown .
paired pre - immune and post - immune sera from these subjects were tested in the passive protection assay , and survival after 14  days is shown .
delay to death was assessed by survival distribution functions using the product - limit approach and compared between groups of mice by log - rank test using proc lifetest in sas version 9.13 .
differences in protection were compared by one - sided fisher 's exact test using proc freq with the exact option in sas version 9.13 . finally , we performed ligand competition experiments to investigate the specificity of the vaccine - induced activity . for these experiments
, we selected 5 serum pairs ( subject nos . 4 , 21 , 37 , 43 , and 61 ) that spanned the different vaccine doses and functional activities .
4 and 61 were selected because the post - immune sera significantly increased survival at day 14 compared to the pre - immune sera , and sera from subject nos .
37 and 43 were selected because the post - immune sera increased survival at day 14 and delayed death compared to the pre - immune serum .
21 because the post - immune serum had weak or no functional activity , as indicated by a non - significant delay to death ( p = 0.06 ) and small increase ( 79.9  eu / ml ) in anti - phtd titer compared to the pre - immune serum . prior to testing in the passive protection model
, the selected serum pairs were incubated for 1  h with recombinant phtd to block phtd - binding antibodies . in parallel reactions
, sera were incubated with lytb , another s. pneumoniae surface protein , as a control .
compared to the pre - immune sera , post - immune sera from all subjects except no .
21 significantly increased mouse survival when incubated with lytb , whereas survival was not significantly increased by any of the post - immune sera incubated with phtd ( table 2 ) .
therefore , most or all of the protective activity in the post - immune sera was due to anti - phtd antibodies . in summary , our results show that the candidate phtd vaccine induces anti - phtd antibodies that can protect against s. pneumoniae infection . another phtd candidate vaccine , adjuvanted with as02v , was recently reported to be immunogenic and well tolerated in adults and to induce antibodies that passively protect mice against a lethal dose of s. pneumoniae .
vaccination with the as02v - adjuvanted candidate vaccine increased the protective activity in sera from older but not younger adults .
the authors concluded that the difference between the 2 age groups was due to higher baseline activity in the younger adults . in the current study , we tried to avoid interference from such baseline functional activity by testing serum dilutions at which the corresponding pre - immune sera was not protective .
in addition , we performed competition experiments to confirm that the functional activity in the sera was specifically due to anti - phtd antibodies and not to antibodies to other pneumococcal antigens , which could have arisen from a s. pneumoniae infection during the trial . 

table 2.specificity of vaccine - induced antibodysurvival , n ( % ) + lytb+ phtdsubjectdilution testedpre - immunepost - immunep - valuepre - immunepost - immunep - value41:204 ( 26.7)11 ( 73.3)0.0131 ( 6.7)3 ( 20.0)ns211:200 ( 0)1 ( 6.7)ns1 ( 6.7)0 ( 0.0)ns371:201 ( 6.7)14 ( 93.3)<0.0010 ( 0.0)0 ( 0.0)ns431:401 ( 6.7)7 ( 46.7)0.0180 ( 0.0)2 ( 13.3)ns611:400 ( 0.0)4 ( 26.7)0.04980 ( 0.0)0 ( 0.0)nspaired pre- and post - immune sera from subject nos .
4 , 21 , 37 , 43 , and 61 were incubated at the indicated dilutions with 20  g / ml phtd or lytb proteins for 1  h. the sera ( 200  l ) were then administered by intraperitoneal injection to 6- to 8-week - old female nave cba / n mice ( n = 15/group ) .
after 1  h , the mice were challenged intravenously with a lethal dose ( 50  cfu in 200  l ) of s. pneumoniae strain a66.1 ( serotype 3 ) , and survival was monitored for 14  days .
p - values were calculated using a one - sided fisher 's exact test comparing survival in mice injected with matched post - immune and pre - immune sera in sas version 8.2 .
specificity of vaccine - induced antibody paired pre- and post - immune sera from subject nos . 4 , 21 , 37 , 43 , and 61 were incubated at the indicated dilutions with 20  g / ml phtd or lytb proteins for 1  h. the sera ( 200  l ) were then administered by intraperitoneal injection to 6- to 8-week - old female nave cba / n mice ( n = 15/group ) .
after 1  h , the mice were challenged intravenously with a lethal dose ( 50  cfu in 200  l ) of s. pneumoniae strain a66.1 ( serotype 3 ) , and survival was monitored for 14  days .
p - values were calculated using a one - sided fisher 's exact test comparing survival in mice injected with matched post - immune and pre - immune sera in sas version 8.2 .
ns , not significant ( p  0.05 ) . according to logistic analysis , naturally acquired and vaccine - induced antibodies had overlapping activities in the passive protection model ( ed50 = 1679  eu / ml [ 95% ci , 14201946 ] for naturally acquired antibodies vs. 1331  eu / ml [ 95% ci , 7622038 ] for vaccine - induced antibodies ) .
another study also found that naturally acquired anti - phtd antibody from human serum can protect mice against a lethal s. pneumoniae intranasal challenge ; however , the study did not examine the relationship between functional activity and vaccination , antibody quantity , or antibody quality .
accordingly , our current results indicate that the candidate phtd vaccine increases functional activity , at least in part , by increasing the antibody concentration .
our preliminary epitope mapping studies and experiments measuring serum binding to bacterial surfaces suggest that vaccination can also expand the epitope repertoire of anti - phtd antibodies ( data not shown ) .
collectively , these results showed that human anti - phtd antibodies , whether naturally acquired or induced by the phtd candidate vaccine , are functional .
this supports the development of the phtd candidate as part of a broadly protective pneumococcal vaccine .
 currently marketed streptococcus pneumoniae vaccines are based on polysaccharide capsular antigens from the most common strains . 
 pneumococcal histidine triad protein d ( phtd ) is a conserved surface protein that is being evaluated as a candidate for a vaccine with improved serotype coverage . here , 
 we measured the functional activity of human anti - phtd antibodies in a passive protection model wherein mice were challenged with a lethal dose of s. pneumoniae by intravenous injection . 
 this functional activity was compared with anti - phtd antibody concentrations measured by enzyme - linked immunosorbent assay ( elisa ) to estimate the 50% protective dose ( ed50 ) . 
 anti - phtd antibodies affinity purified from pooled normal human sera passively protected mice with an ed50 of 1679 elisa units / ml ( 95% confidence interval , 14201946 ) . 
 sera from subjects injected with aluminum - adjuvanted phtd in a phase i trial had similar activity per unit of antibody ( ed50 = 1331 elisa units / ml [ 95% confidence interval , 7622038 ] ) . 
 vaccine - induced activity in the passive protection model was blocked by pre - incubation with recombinant phtd but not by a control s. pneumoniae antigen ( lytb ) . 
 these results show that human anti - phtd antibodies , whether naturally acquired or induced by the phtd candidate vaccine , are functional . 
 this supports the development of the phtd candidate as part of a broadly protective pneumococcal vaccine . 
Abbreviations
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