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over several decades , the pharmaceutical industry has become heavily reliant on demonstrating functionality in animal models , especially mouse models , as a way to characterize and select vaccine formulations .
however , it is becoming increasingly clear that animal testing may not always accurately reflect the response in humans and may not necessarily provide the most meaningful data .
the issues surrounding animal use and human immunofunctionality are well - known and have been highlighted in several recent publications .
the difference in immune function is hardly surprising given the vast evolutionary distance between mouse and human , thought to be in the order of 65 million years .
there are many examples where differences between animals and humans may have profound implications for formulation development .
one example is toll - like receptor ( tlr ) usage , relevant to the development of new generation adjuvants .
currently , the response initiated by a tlr may not be fully known until clinical testing is initiated .
another example was highlighted in a clinical trial for an immunomodulatory anti - cd28 monoclonal antibody ( mab ) ( tgn1412 ) . during the trial , delivery of the antibody triggered an immediate systemic inflammatory response and
a serious adverse event ( sae ) referred to as a  cytokine storm . 
this life - threatening event was unexpected since standard non - clinical safety studies in animal models raised no alarm . during pharmaceutical development of vaccines , it is crucial to identify optimal conditions for long - term storage and for boosting immunogenicity through , for example , the addition of adjuvants . to this end ,
a large number of variables must be examined during pre - formulation development to identify optimal ph , buffer ingredients , ionic strength , stabilizer excipients , as well as optimal adjuvant - antigen composition .
multiwell - based assays are readily available to monitor biophysical and biochemical properties of macromolecules as a function of different formulation parameters under normal or stressed conditions .
however , there is a lack of simple and cost efficient in vitro high throughput screening methods to evaluate the biological activity of complete adjuvanted formulations .
the hwb approach aims to emulate this . in order to begin addressing some of the issues
mentioned above and to bridge preclinical testing in animal models , sanofi pasteur has looked into strategies that focus on in vitro testing using human cells .
compatible with these initiatives and as a way to further bridge formulation development to vaccine functionality and to human clinical studies , we now describe a new way to evaluate the immune response to adjuvanted formulations using a fresh human whole blood ( hwb ) approach ( fig .  1 )
. the approach may be particularly useful in an early clinical phase when a formal in vitro potency assay may not be available .
innate immunity is an immediate response to a stimulus or  danger signal  typically triggered through ancient receptors inherited at birth , which sense conserved pathogen - associated molecular patterns ( pamps ) found on a variety of bacteria , fungi , and other pathogens .
one of the best characterized of these innate signaling pathways is that for the bacterial endotoxin , lipopolysaccharide ( lps ) , which triggers a pro - inflammatory response through toll - like receptor 4 ( tlr4 ) .
derivatives of lps that mimic innate immunity are now key components of a new generation of vaccine adjuvants that use a tlr4 agonist ( tlr4a ) .
studies were initially conducted using tlr4a combined with an aluminum salt adjuvant ( asa ) , hereafter referred to as tlr4-adjuvant .
several of these adjuvant formulations stimulated a pro - inflammatory cytokine ( tnf ) response in fresh human whole blood ( hwb ) .
the response was initiated after as little as 6 h of incubation and more optimally between 1824 h. to further characterize the adjuvant response , individual components of the adjuvant were used in a follow - up study to stimulate hwb over a broad range of concentrations .
the data indicated a consistent trend in response across 3 different subjects ( fig .  2 ) .
although there was a lack of response to the asa alone ( not shown ) , the response to tlr4a was significantly augmented in the presence of asa . indeed , there were responses to the tlr4-adjuvant at concentrations of the tlr4a alone that would otherwise have failed to induce a detectable response .
tnf is released from human whole blood of three different subjects after 24h stimulation with lps , tlr4-adjuvant , tlr4a , and asa ( asa alone was consistently negative and is not shown ) . while there is expected variability between subjects , there may be ways to normalize the data against a standard reference such as lps or other tlr4as .
evaluating adjuvant responses in this way is inexpensive , relatively quick , and technically easy to perform .
depending on the technology used it may be possible to generate functional data comfortably within a single working day .
typically , the memory t cell population is rapidly activated and proliferates upon re - encounter with a specific antigen , with immediate cytokine - secreting cells being described as effector memory , and with proliferating t cells being described as central memory .
a methodology developed by scientists at the london school of hygiene and tropical medicine and based on earlier studies in leishmania has been in use for approximately two decades as a way to monitor antigen specific , vaccine - induced memory after bacillus calmette - gurin ( bcg ) vaccination .
the response is monitored by stimulating hwb with antigens from m. tuberculosis and detection of t cell cytokines such as ifn after 67 d of culture by elisa , or by multiplex bead array assays . to investigate antigen specific responses to complete vaccine formulations , an advanced tb vaccine candidate referred to as h4 was formulated with the tlr4-adjuvant and tested using fresh hwb recovered from 3 healthy bcg vaccinated volunteers ( fig . 
the data demonstrated that an antigen - specific ifn memory response can be monitored in a simple 6 d assay when blood is incubated with the complete formulation . in all cases
the optimum concentration of h4 was relatively broad ( 2.0 g and 0.02 g ) and was dependent on the concentration of tlr4-adjuvant .
while responses were variable between subjects , the expectation is that loss of potency , between different formulations , would be apparent in any subject tested .
the ability to demonstrate profound immunomodulation in terms of quantitative ( measurable ) and qualitative ( cytokine profile ) responses by adjuvant on the t cell response through this in vitro approach is of particular interest .
the use of hwb may provide a new way to monitor  complete vaccine formulations in terms of both the antigen and the adjuvant response .
the ability to demonstrate and understand modulation in vitro in this manner is relatively novel . since
a tlr4-adjuvant is expected to drive th1-like immunity , it is thus important to show and quantify this effect for any formulation being developed .
the example chosen here uses a tb vaccine to boost the memory response primed by bcg or prior mycobacterial exposure .
however , the approach is likely to be applicable to any other t cell inducing vaccine that aims to boost existing memory .
once the functionality of a formulation can be demonstrated , the ability to detect changes to the stability of the formulation is then possible . to this end ,
tlr4-adjuvant formulations under different conditions of accelerated temperature storage were monitored using hwb and chemical integrity by reversed phase high - performance liquid chromatography ( rp - hplc ) .
the fresh blood from a single subject was tested on multiple weeks using different adjuvant preparations in an overnight tnf assay .
the data revealed a loss of functional activity ( ability to stimulate tnf ) when formulations were stored at 45 c and 60 c for 3 , 5 , and 12 wk ( fig . 
interestingly , the loss of functional activity agreed with changes in the rp - hplc profile .
the ability to link specific chemical or structural changes to an immunofunctionality will be particularly useful .
effect of temperature on chemical degradation and functionality of a tlr4 agonist as measured by rp - hplc ( green ) and in hwb ( purple ) .
since the approach identifies both innate signaling and the modulated adaptive response , the use of cells from healthy humans may be a quick way to confirm absence of unwanted or unexpected immunity in any formulation at any time during development .
such testing might also identify contamination with endotoxin or any other toxins that might not be recognized in other standard tests ( i.e. , limulus amebocyte lysate  lal and rabbit pyrogenicity ) .
indeed , a commercial kit using hwb is already being marketed for this purpose ( biotest pyrodetect system ) .
application of the approach may also be helpful to a non - clinical safety assessment .
certainly , innovative approaches to address safety are of major interest to the industry ( e.g. , biovacsafe ) .
the hwb approach enables the determination of the functionality of a complete formulation ( adjuvant and antigen ) within 6 d. multiple formulations can be monitored simultaneously and responses compared for several subjects .
the approach presents an opportunity for a broad formulation screen and to select optimal , possibly safer , formulations based on a functional response on fresh human cells .
the functional response can be used in conjunction with standard biochemical and biophysical tests . the ability to re - stimulate antigen - specific memory b cells
moreover , the ability to correlate a memory t helper cell response with antibody elicited in vivo would make the approach especially attractive for screening other vaccine targets .
these technologies and other assays such as qpcr or proteomics could be readily applied to the hwb approach .
a convincing argument for the advantages of using fresh hwb is already reported and discussed .
provided that drawn blood is returned to culture within a few hours , there is no need for cellular enrichment or centrifugation .
in addition , hwb contains components essential to stimulate both innate and memory t cell immunity .
such factors may include neutrophils and eosinophils , as well as platelets and other cells and plasma constituents , that have been shown to play a role in cellular function and cytokine production .
since hwb is used for immunomonitoring clinical trials , there may be a direct link between formulation screening in vitro and actual clinical trial monitoring .
indeed , the approach may be compatible with a  trial - in - a - test tube , where an in vitro response may be predictive of the clinical response .
the use of point - of - care devices ( pcd ) to screen whole blood for an array of markers is becoming a reality .
such variability allows for a more realistic understanding of vaccine performance . in terms of formulation screening
, multiple formulations can be monitored relative to a reference formulation within a single subject . in summary ,
the hwb approach is a relatively inexpensive , fast hts methodology that can monitor complete vaccine formulation functionality in terms of the adjuvant response , the antigen response , and the modulation of the antigen response by the adjuvant .
in addition , the approach can detect a loss of functionality thus supporting structure - function characterization of vaccine antigens and optimization of antigen - adjuvant interactions .
finally , the application of a formulation to human cells can be a sensitive indicator of unwanted or unexpected immunological response .
the approach would considerably de - risk progression of vaccine formulations through early clinical trials ( fig .
understanding the relevant biological activity of any pharmaceutical formulation destined for human use is crucial . for vaccine - based formulations , activity must reflect the expected immune response , while for non - vaccine therapeutic agents , such as monoclonal antibodies , a lack of immune response to the formulation is desired . 

 during early formulation development , various biochemical and biophysical characteristics 
 can be monitored in a high - throughput screening ( hts ) format . 
 however , it remains impractical and arguably unethical to screen samples in this way for immunological functionality in animal models . 
 furthermore , data for immunological functionality lag formulation design by months , making it cumbersome to relate back to formulations in real - time . 
 it is also likely that animal testing may not accurately reflect the response in humans . 
 for a more effective formulation screen , a human whole blood ( hwb ) 
 approach can be used to assess immunological functionality . 
 the functional activity relates directly to the human immune response to a complete formulation ( adjuvant / antigen ) and includes adjuvant response , antigen response , adjuvant - modulated antigen response , stability , and potentially safety . 
 
 the following commentary discusses the hwb approach as a valuable new tool to de - risk manufacture , formulation design , and clinical progression .