Biomarkers and assessment of vaccine responses
WAYNE R. HOGREFE
Focus Technologies, Cypress, CA, USA
Vaccines for infectious diseases have in the past, and will into the future, relied on a variety of
surrogate markers to monitor vaccine efficacy. The primary surrogate markers have been either
the antibody titer to vaccine antigens or the measurement of antibody function such as anti-viral
neutralizing activity. In recent years, the measurement of T-cell function in conjunction with or
independent of antibody measurements have been used to assess vaccine efficacy. ELISPOT,
flow cytometry and intra-cellular staining methods are used to determine the impact of vaccines
on immune mediators such as interleukins, interferons, MHC expression and pro-inflammatory
mediators. The relevant B-cell and T-cell surrogate markers for vaccine efficacy is dependent on
the vaccine being used, so that no universal set of surrogate markers can be applied to all
vaccines. The use of T-cell surrogate markers can be complicated by the lack of sensitivity to
accurately measure intra-cellular mediators. Although typically this is not a problem for
infectious disease vaccines, it is a major problem for cancer vaccines.
Keywords: Vaccine response, ELISPOT, tetramer
The term ‘biomarkers’ covers a multitude of chemical and biological molecules in a
wide range of pathologic conditions including infectious disease, cardiovascular
disease, neurological diseases, inflammatory diseases and many more. Recently, the
term biomarker has been defined as biomolecules such as mRNA, proteins, peptides
and small molecules, that can be quantitated, predict disease or drug activity and
are ‘novel’ (Diller 2004). ‘Novel’ in this definition means the biomarker is neither
an established diagnostic marker nor a surrogate marker used to predict clinical
endpoints. With such a strict definition, no markers used in infectious disease and
cancer vaccine development would qualify as a ‘biomarker’, since the only vaccine
markers available are surrogate markers measuring various components of the
immune system. Therefore, this review is limited to surrogate markers of the immune
response to both infectious disease and cancer vaccines.
Biomarkers are utilized at two stages of vaccine development. The first stage is to
identify appropriate immunogens where the measurement of end-stage immune
function such as antibody levels is utilized. The second stage is to establish vaccine
efficacy as well as assess protection of the proposed vaccine (Krause 1998). Both
humoral and cell-mediated immunity biomarkers are used in this stage. Immune
response biomarkers that correlate with protection are used to determine efficacy
Correspondence: Wayne R. Hogrefe, 5785 Corporate Avenue, Cypress, CA 90630, USA. Tel: 714-220-
1900. Fax: 714-821-3364. Email: firstname.lastname@example.org
ISSN 1354-750X print/ISSN 1366-5804 online # 2005 Taylor & Francis
Biomarkers, November 2005; 10(Supplement 1): S50?/S57
issues such as the proper vaccine formulation, inoculation schedules, dosage and
target populations (Siber et al. 1998). When measuring protection of potential new
vaccines, it must be kept in mind that cancer and infectious disease vaccines have
different roles in patient protection and, thus, different markers will be used to assess
protection. For infectious disease vaccines, the primary goal is the protection of the
general population from acquiring disease, while for cancer vaccines protection of the
individual patient from residual disease is the primary goal (Ka ¨yhty 1998, Lyerly et al.
2001, Weiner & Kim 2002). Due to these different primary roles and the different
immune-mediated protection that is required, infectious disease vaccine protection is
measured primarily with humoral markers with cellular immune markers being
secondary. In cancer vaccines, the opposite has occurred where cell-mediated immune
biomarkers are the primary measures used (Lyerly et al. 2001).
Traditional surrogate markers for vaccine evaluation
Surrogate markers for immunity have been in existence for nearly 150 years. In the
19thcentury, Robert Koch described the use of the skin test (injection of inactivated
virus intra-dermally) to indicate the host’s delayed-type hypersensitivity to poxviruses.
The skin test became the first surrogate marker to indicate the presence of immunity
to poxviruses and, thus, an indicator of protection. Since the 19thcentury, a number
of surrogate markers have been used to assess immunity and, in particular, to assess
the immune response to vaccines. Besides the traditional skin test, the cell-mediated
arm of the immune response has been measured by quantitating the in vitro
proliferation of mononuclear cells, primarily lymphocytes, when these cells are placed
in culture with the particular antigen being assessed. The proliferation of lymphocytes
has traditionally used the incorporation of
quantitative measure of memory cells to the antigen being assessed. A second in vitro
measurement of cell mediated immunity, especially in cancer and viral immunity, is
the quantitation of killer cell function mediated by lymphocytes previously sensitized
to the antigens present on the target cells. Target cells are labelled with soluble intra-
cellular compounds such as57chromium that are released into the culture medium
upon cell lysis. The number of labelled target cells lysed by cytotoxic T-cells is a
measure of immunologic memory to the target antigens in question whether they are
cancer antigens or viral antigens expressed on the surface of the target cells.
The measurement of antibodies has been the most widely used surrogate marker of
immunity. The most common antibody measure is quantitating the level of antibody
present in serum to a particular antigen or pathogen. The quantity of antibodies in
serum to such pathogens as diphtheria and tetanus has long been used to determine if
an individual has sufficient levels of pathogen-specific antibodies to be protected from
subsequent exposure to that pathogen. The functional activity of antibodies is also
measured to determine efficacy of vaccines (Ka ¨yhty 1998). Examples of functional
measures of humoral immunity include bacterialcidal assays (H. influenza type b,
meningococcus), opsonophagocytic activity (S. pneumoniae, H. influenza type b),
toxin or virus neutralization (tetanus, diphtheria, poliovirus) and IgG avidity (a
measure of immunologic memory). In the latter example, the binding avidity of IgG to
its specific antigen has been known to increase over time after the initial exposure of
the host to the pathogen. As shown in Figure 1, Ashley-Morrow et al. (2004)
demonstrated the increased avidity of IgG to the specific antigen of HSV-2 (gG2) over
3H thymidine in dividing cells as a
Biomarkers and assessment of vaccine responses
time after the initial exposure to HSV-2 genital infection. It is now known that
increasing avidity of IgG to a particular antigen is a function of immunologic memory
in the host that is required for long-term protection (Usinger & Lucas 1999, Joseph
et al. 2001).
Although all of the above markers of immune function have existed for decades,
they are still today the primary markers used for infectious disease vaccine efficacy. As
recently as 2003 (Jo ´dar et al. 2003), published criteria that should be used to assess
the next generation of conjugated pneumococcal vaccines. The primary criteria is
the attainment of threshold levels of protective antibodies, the secondary criteria is the
measurement of functional antibodies using the opsonophagocytic assay and the
tertiary criteria was the measurement of immunologic memory by either measuring
antibody avidity or detecting increasing antibody levels with booster injections.
Next generation of biomarkers for vaccines
The traditional biomarkers outlined above measure the final immunologic outcome or
protective capability of vaccines. As knowledge of immunologic mechanisms expands,
the next generation of vaccine biomarkers allows one to measure vaccine efficacy at
the individual immune cell level rather than measuring the total immune response.
The new biomarkers almost exclusively measure T-cell function at the memory, helper
and effector levels, as well as T-cell interaction with other cells of the immune system
such as dendritic or antigen presenting cells. These biomarkers are designed to attain
information at the individual immune cell level, incorporate individual cell phenotypic
information together with cell function, assess memory and function at the T-cell
0-3 wks3-6 wks 6wks - 3mo 3-6 mo 6mo - 5yrs > 5 yrs
n = 46
31.4 31.843.7 60.7 76.394.073.676.0 median =
Figure 1. HSV-2 IgG avidity as a function of time after the primary genital herpes episode (Ashley Morrow
et al. 2004).
W . R. Hogrefe
receptor level and measure function via cytokine production at the intra-and extra-
cellular levels (Elahi et al. 2003, Gans et al. 2003, Ovsyannikova et al. 2003).
Figure 2 is a very simplified schematic representation of the immune system where
the T-cell has a central role in both regulatory and effector functions of the immune
response. The immune response is controlled by numerous soluble factors and
immune response mediators such as tumour necrosis factor (TNF-a), interferon-
gamma and numerous interleukins (IL). Described below are three methods that
either measure cell function via the measurement of the soluble immune factors
(cytokines) at the individual cell level or monitor cell receptor-antigen receptor
interaction. The methods include the binding of tetramers to cell surface receptors,
measurement of epitope immunoreactivity at the individual cell level using the
ELISPOT and the simultaneous measurement of intra-cellular cytokine production
and cell phenotype using flow cytometry.
Figure 3 illustrates the interaction between dendritic cells and T-cells where the
dendritic cell is responsible for presenting to the T-cell the specific antigen (epitope) in
a proper orientation so as to be recognized the T-cell receptor (TCR). The proper
antigen orientation includes not only the epitope itself but also the appropriate major
histocompatibility complex (MHC) molecules and other cell surface molecules such
as CD80/86. T-cells possess a large repertoire of TCR and the presentation of the
correct epitope/MHC/CD molecules that ‘fit’ into the TCR will initiate the
subsequent immune cascade leading to the development of T-cell memory, Th1 or
Th2 regulatory cells or some other effector cell function. For vaccines, therefore,
Figure 2. Schematic of T-cell interaction.
Biomarkers and assessment of vaccine responses
finding the best epitope to stimulate the desired immune response is critical.
Tetramers are synthetic versions of the dendritic cells’ antigen presentation apparatus
including the MHC molecules. Tetramers (Figure 4) are four single synthetic
peptides/MHC complexes bound by a ringed structure (beta-2 microglobulin) to
form a ‘four-plex’ or tetramer. The four-plex is more efficient and has a higher avidity
than a single-plex molecule to bind to TCR in vitro. The tetramer also incorporates a
fluorescent label so that when the tetramer binds to a T cell receptor in vitro, the
binding event can be measured by flow cytometry.
Tetramers have several uses, but typically they are used identify the peptide
sequence or epitopes that bind to the highest number of TCR in a naı ¨ve individual as
well as identifying the phenotype of the T-cell to which the tetramer binds. Tetramers
Figure 3. The interaction of dendritic cells and T-cells involving the T-cell receptor (TCR), major
histocompatibility complex (MHC) antigens and inter-cellular signalling molecules.
Figure 4. Structure of tetramer. Monomer consists of a synthetic molecule containing a peptide
representing an epitope of interest, MHC molecule and beta-2 microglobulin.
W . R. Hogrefe
can also measure the changes in the number of T-cells displaying a particular TCR
before and after vaccination. Tetramers are now synthesized with both Class I and
Class II MHC molecules so that binding to either Th1 or Th2 T-cells, respectively,
can now be accomplished. Since tetramer binding is measured in a flow cytometer, the
phenotype of the T-cell can be identified by the addition of monoclonal antibodies
containing a fluorescent tag different than that present on the tetramer. The limit of
detection of tetramer binding T-cells is one tetramer-binding cell per 8000 cells. After
vaccination, a 2?/5-fold increase in tetramer-binding cells typically occurs. Tetramers
have been used for both infectious disease applications as well as identifying cancer
vaccine candidates for melanoma, leukaemia, lymphoma and other cancers. Although
tetramers are useful, the results can be difficult to interpret, since these measurements
require high precision instrumentation due to the rare event analysis that is required
to detect cell binding tetramers (Hoffman et al. 2000, Terajima et al. 2003, Whiteside
et al. 2003, Walker et al. 2004, Ye et al. 2004).
In the 1990s, the ELISPOT technique was standardized and shown to correlate
with the standard assay of the day to measure T-cell effector function, namely the
chromium-release cell-mediated cytotoxicity assay. The ELISPOT gave the added
benefit that it could measure individual soluble factors such as interferon-gamma, IL-
4, IL-10 and TNF-a that are secreted by T-cells in response to an immunologic
stimuli, i.e. measuring post-vaccine immune responses (Arlen et al. 2003). The
ELISPOT technique utilizes 96 well micro-titer plates coated with a monoclonal
antibody to the specific soluble factor being measured (INF-gamma, IL-4, etc.) where
it functions as a capture antibody. The lymphoid cell population being investigated is
then added to the microtiter wells containing the captured antibodies in a limiting
dilution pattern. The specific antigen is also added to the wells and the cells are
cultured for 6?/48 hours. The cells and antigen are then washed from the well and
replaced with a second monoclonal antibody containing an enzyme label to the
specific soluble factor being measured. By developing the microtiter plate with the
appropriate chromogen, spots will appear in the bottom of the microtiter well
wherever a cell was present in the primary culture that secreted the soluble factor
being measured. Since the number of cells originally added to the micro-titer well is
known, it is possible to quantitate the number of cells responding to the antigen
present in the cell population being studied.
As shown in Figure 2, measuring different soluble factors allows the investigator to
assess different pathways of the immune system when activated by various vaccine
regimes. For example, IFN-gamma is often measured to quantitate the number of
activated T-cells present in the cell population being investigated. ELISPOT, like
tetramer binding studies, is capable of identifying the presence of antigen reactive cells
in very low concentration, as low as one cell in 50000 (Terajima et al. 2003, Whiteside
et al. 2003, Hudgens et al. 2004). Since ELISPOT reagents are now commercially
available, this technique to dissect the immune response at the individual cell level is
now a common practice.
The final method to assess immune reactivity to vaccines at the individual cell level
is cytokine flow cytometry (CFC). CFC is similar to ELISPOT in that it allows one to
measure the generation of specific cytokines in response to a particular antigen
stimulus; however, CFC will also identify the phenotype of the cell stimulated to
produce intra-cellular cytokines. Briefly, lymphoid cells are stimulated by a specific
antigen in short-term cultures, usually less than 24 hours. After the short-term
Biomarkers and assessment of vaccine responses
cultures, the presence of intra-cellular cytokines is detected by fluorescein-labelled
monoclonal antibodies. A second set of monoclonal antibodies with different
fluorescein tags are used to identify the phenotype of the lymphoid cell stimulated
to produce the intra-cellular cytokines. Although CFC does not measure cell function,
it is very sensitive in detecting antigen reactive cells with a limit of detection of the one
cell in 50000. Thus, CFC can detect the presence of various T-cell sub-populations
that will respond to a vaccine stimulus. This information can be used to modify both
antigens and adjuvants to stimulate the T-cell populations desired, such as having an
adjuvant that favours the stimulation of Th1 rather than Th2 cells (Whiteside et al.
2003, Mangada et al. 2004)
Practical applications of vaccine biomarkers
Recently, Wang et al. (2004) used nearly all the biomarkers available to develop a
malaria vaccine strategy combining recombinant antigens and DNA vaccines. The
investigators used the newer techniques such as IFN-gamma ELISPOT and
tetramers, together with the traditional biomarkers of P. falciparum antibody levels
and cell mediated cytotoxicity assays to investigate each arm of the vaccine strategy.
They found the recombinant protein vaccines produced antibodies, but only short-
term protection and no IFN-gamma producing cells. The DNA vaccine given
separately did not give protection or tetramer-binding T-cells, although cytotoxic T-
cells were detected. Using this biomarker information they developed a combined
recombinant protein/DNA vaccine strategy that produced antibody, IFN-gamma
producing cells, cytotoxic T-cells and increased protection.
Finally, Whiteside and Gooding (2003) recently reviewed the use of biomarkers to
monitor the efficacy of gene therapy to enhance the immune response to residual
disease in leukaemia and lymphoma patients. They found that cell population-based
assays such as cytotoxicity and proliferation assays were not sensitive enough to be
used to measure cancer vaccine or gene therapy efficacy. They also found that the
single cell based assays such as tetramer binding, ELISPOT and CFC lack
standardization and typically did not correlate with each other. Even though these
single cell assays were able to detect reactive cells at levels of less than one cell per
10000, this level of sensitivity was still not useful for cancer vaccine studies. For their
studies in cancer gene therapy, they recommend the use of skin testing with
appropriate cancer cell derived antigens to measure therapy efficacy. One hundred
and fifty years later, Dr Koch would be proud.
Arlen PM, Gulley JL, Palena C, Marshall J, Schlom J, Tsang KY. 2003. A novel ELISPOTassay to enhance
detection of antigen-specific T cells expressing vector-driven human B7-1. Journal of Immunology
Ashley Morrow R, Friedrich D, Krantz E, Wald A. 2004. Development and utility of a type specific antibody
avidity test based on Herpes Simplex virus type 2 glycoprotein G. Sexually Transmitted Diseases 31:508?/
Diller W. 2004. Roche’s challenging biomarker strategy. In vivo: The Business and Medicine Report, 22
Elahi S, Pang G, Clancy R. 2003. Development of surrogate markers for oral immunizations against
Candida albicans. Vaccine 21:671?/677.
W . R. Hogrefe
Gans H, DeHovitz R, Forghani B, Beeler J, Maldonado Y, Arvin AM. 2003. Measles and mumps
vaccination as a model to investigate the developing immune system: passive and active immunity during
the first year of life. Vaccine 21:3398?/3405.
Hoffmann TK, Donnenberg VS, Friebe-Hoffmann U, Meyer EM, Rinaldo CR, DeLeo AB, Whiteside TL,
Donnenberg AD. 2000. Competition of peptide-MHC class I tetrameric complexes with anti-CD3
provides evidence for specificity of peptide binding to the TCR complex. Cytometry 41:321?/328.
Hudgens MG, Self SG, Chiu YL, Russell ND, Horton H, McElrath MJ. 2004. Statistical considerations for
the design and analysis of the ELIspot assay in HIV-1 vaccine trials. Journal of Immunology Methods
Jo ´dar L, Butler J, Carlone G, Dagan R, Goldblatt D, Ka ¨yhty H, Klugman K, Plikaytis B, Sibor G,
Kohberger R, Chang I, Cherian T. 2003. Serological criteria for evaluation and licensure of new
pneumococcal conjugate vaccine formulations for use in infants. Vaccine 21:3265?/3272.
Joseph H, Miller E, Dawson M, Andrews N, Feavers I, Borrow R. 2001. Meningococcal serogroup A avidity
indices as a surrogate marker of priming for the induction of immunologic memory after vaccination with
a meningococcal A/C conjugate vaccine in infants in the United Kingdom. Journal of Infectious Diseases
Ka ¨yhty H. 1998. Immunogenicity assays and surrogate markers to predict vaccine efficacy. In: Plotkin S,
Brown F, Horaud F, editors. Preclinical and clinical development of new vaccines. Developments in
Biological Standards 95. p. 175?/180.
Krause DD. 1998. Introduction to surrogate markers. Annual Conference on Vaccine Research; 30 May.
p. 26 (abstract no. S5).
Lyerly HK, Morse MA, Clay TM. 2001. Surrogate markers of effective anti-tumor immunity. Annals of
Surgical Oncology 8:190?/191.
Mangada MM, Ennis FA, Rothman AL. 2004. Quantitation of dengue virus specific CD4? / T cells by
intracellular cytokine staining. Journal of Immunology Methods 284:89?/97.
Ovsyannikova IA, Reid KC, Jacobson RM, Oberg AL, Klee GG, Poland GA. 2003. Cytokine production
patterns and antibody response to measles vaccine. Vaccine 21:3945?/3953.
Siber GG, Kohberger R, Xie F. 1998. Surrogate markers, lessons from the past. Annual Conference in
Vaccine Research; 30 May (abstract S6).
Terajima M, Cruz J, Raines G, Kilpatrick ED, Kennedy JS, Rothman AL, Ennis FA. 2003. Quantitation of
CD8? / T cells to newly identified HLA-A*0201-restricted T cell epitopes conserved among vaccinia and
variola (smallpox) viruses. Journal of Experimental Medicine 197:927?/932.
Usinger WR, Lucas AH. 1999. Avidity as a determinant of the protective efficacy of human antibodies to
pneumococcal capsular polysaccharides. Infectious Immunology 67:2366?/2367.
Walker EB, Haley D, Miller W, Floyd K, Wisner KP, Sanjuan N, Maecker H, Romero P, Hu HM, Alvord
WG, Smith JW, Fox BA, Urba WJ. 2004. gp100 (209-2M) peptide immunization of human lymphocyte
antigen-2A? / stage I-III melanoma patients induces significant increase in antigen-specific effector and
long-term memory CD8? / T cells. Clinical Cancer Research 10:668?/680.
Wang R, Epstein J, Charoenvit Y, Baraceros FM, Rahardjo N, Gay T, Banania JG, Chattopadhyay R, de la
Vega P, Richie TL, Torneiparth N, Doolen DL, Kester KE, Heppner DG, Norman J, Carucci DJ, Cohen
JD, Hoffman SL. 2004. Induction in humans of CD8? / and CD4? / T cells and antibody responses by
sequential immunization with malaria DNA and recombinant proteins. Journal of Immunology
Weiner DB, Kim JJ. 2002. Cancer vaccines: is the future now? Expert Reviews in Vaccines 1:257?/260.
Whiteside TL, Gooding W. 2003. Immune monitoring of human gene therapy trials: potential application to
leukemia and lymphoma. Blood Cells, Molecules and Diseases 31:63?/71.
Whiteside TL, Zhao Y, Tsukishiro T, Elder EM, Gooding W, Baar J. 2003. Enzyme-linked immunospot,
cytokine flow cytometer, and tetramers in the detection of T-cell responses to a dendritic cell-based
multipeptide vaccine in and patients with melanoma. Clinical Cancer Research 9:641?/649.
Ye M, Kasey S, Khurana S, Nguyen NT, Shubert S, Nugent CT, Kuus-Reichel K, Hampl J. 2004. MHC
class II tetramers containing influenza hemagglutinin and EBV EBNA1 epitopes detect reliably specific
CD4(? /) T cells in healthy volunteers. Human Immunology 65:507?/513.
Biomarkers and assessment of vaccine responses