|DNA vaccines for prophylaxis and therapy
DNA vaccines are tatnalizingly
giving hope of a third vaccine revolution. Ever since the serendipitous discovery of Wolff
et al.1 that naked DNA injection into the muscle of mice led to
expression of the encoded marker protein, there has been a surge to use this approach to
generate DNA vaccines against a variety of infectious diseases. While, many aspects of the
basic of genetic immunization need to be worked out, some unique features of this
phenomenon have become evident. The injected plasmid DNA with appropriate promoter and
regulatory sequences remains as an episome, but is transcribed and translated to give rise
to the vector-encoded protein. This protein is processed similar to an antigen presented
by an infectious virus, resulting in presentation of antigenic fragments in association
with MHC-class I determinants, leading to activation of cytolytic cells. The generation of
CD8(+) CTLs is proving to be crucial f or defense against many viral, bacterial and
parasitic diseases. Genetic immunization also results in stimulation of T helper cells and
B cells leading to antibody production, which is generally weak, but long investigations.
Since, muscle cells express only low levels of MHC-class I antigens and lack expression of
MHC-class II antigens, it has been suggested that dendritic cells in muscle tissue could
get activated and up-regulate MHC and co-stimulatory molecules. These start to secrete
cytokines, migrate to lymphatic tissues and initiate immune response. Since, the dendritic
cells have a finite life span, the muscle cells being poor target for CTLs, might serve as
a reservoir of antigen, providing a constant reminder to the immune system2.
While, more research is needed to reconcile a wide
variety of immune responses to DNA vaccines described in literature, the dominant CTL
response that is proving to be crucial for protection against major diseases such as
malaria, AIDs and tuberculosis has led to high hopes on this approach for protection.
Besides, DNA vaccine does not need an external adjuvant, sequences such as the CpG motif
in the DNA providing this effect. Finding an appropriate adjuvant for the human, besides
Alum, has been a major effort.
In the case of malaria, studies with a whole gamut
of antigens defining the pre-erythrocytic/liver, erythrocytic and mosquito stages, have
indicated that induction of CD8(+) T cells is crucial and this would have to include
multiple epitopes from single and many proteins, because of parasite polymorphism and
genetic restriction of T cell response. One approach has been to stitch different epitopes
and make a synthetic protein by recombinant route. Spf66 was the first recognized malaria
vaccine developed by Patarroyo by joining three merozoite derived proteins with repetitive
sequences derived from the circumsporozoite protein of P. falciparum. Despite the
initial claims, this vaccine has given equivocal results on human trials in more than one
location3. The latest in this effort is the development of a recombinant
multistage P. falciparum candidate vaccine through an Indo-US collaboration4.
A 41 kDa protein has been expressed in the Baculovirus system from a synthetic gene
consisting of 12B cell, 6 T cell proliferative and 3 CTL epitopes derived from 9
stage specific P. falciparum antigens, corresponding to sporozoite, liver,
erythrocytic asexual and sexual stages. The candidate vaccine remains to be tested in
animal models. One problem with such vaccines could be that the synthetic protein would
fold and only expose certain epitopes but not others that are necessary. A vaccine with a
mixture of several recombinant antigens would prove to be expensive, besides the effect of
protein protein interaction in vitro being anybodys guess.
In this context, DNA vaccines have distinct advantage, where plasmid DNAs encoding
different antigens and prepared by the same generic procedure can be mixed and
administered. A mixture of 4 plasmid DNAs (pfCSP, pfSSP2, pf EXP-1 and pfLSA-1) has been
injected into Rhesus monkeys and found to elicit multiple antigen-specific CTLs5.
There is information that mixtures of 1520 plasmid DNAs are being tested. Infact,
Hoffman et al.6 have suggested a vaccinome approach to make
a pre-erythrocytic vaccine. The scheme envisages construction of DNA vaccine plasmids from
each of thousands of identified open reading frames in the parasite genome an d used to
immunize mice. Antisera from each immunized group is then used to identify the proteins
expressed in irradiated sporozoite infected hepatocytes. (Despite all the
advances, the best result on protection against malaria has only been achieved by
injecting irradiated sporozoites, which ofcourse is not a practical proposition.) From the
protein sequences of hundreds of expressed proteins, the full complement of degenerate HLA
superfamily binding motifs are selected and validated experimentally. The DNA sequences of
selected T-cell epitopes are linked on numerous plasmids giving rise to a vaccine
comprised of tens to hundreds of DNA vaccine plasmids, each containing dozens of
individual T-cell epitopes. A perfect example of mega science!
The importance of CTL response in HIV patients has
also been highlighted. During the symptom-free stage, multiple HIV-1 epitope specific CTLs
can be detected in peripheral blood. This activity decreases with the onset of symptoms.
Several plasmid borne HIV genes have been tested in experimental animals. A recent study
has investigated the immunogenicity of DNA constructs of the regulatory HIV genes, nef,
rev and tat, in human beings7. These three regulatory genes are expressed early
in the life cycle of the virus, and, therefore, it is possible that an immune response to
these genes can eliminate the infected cells before the release of viral particles. The
study carried out with 9 symptom-free, HIV infected patients indicates that DNA
vaccination induced detectable memory cell in all patients and MHC-class I restricted CTLs
of CD8(+) origin in 8 patients. While, the study cannot be generalized in view of the
small number of patients used, it indicates the possibility that a combination of DNA
constructs that encode the HIV-1 regulatory genes might induce a full immune response in
individuals who are already infected. In addition to being a therapeutic vaccine, it might
also work as a prophylactic vaccine in non-infected individuals.
Yet another option is to examine whether DNA vaccine
can serve as an adjunct to chemotherapy. This approach would be very valuable both in AIDS
and tuberculosis, for which satisfactory vaccines are still not available. The only
vaccine available against tuberculosis is BCG with all its limitations. An effective
chemotherapy against tuberculosis is available, but it involves treatment with large doses
of drugs for atleast 6 months after diagnosis. In a recent study8, it has been
shown that a DNA vaccine coding for a mycobacterial heat shock protein of Mr 65000 (Hsp
65), when administered in 4 doses to mice 8 weeks after intravenous injection of virulent M.
tuberculosis H37RV, leads to a dramatic decrease in the numbers of live bacteria in
spleen and lungs 2 months and 5 months after the first dose of DNA. Certain other
mycobacterial antigens and BCG did not have this effect. The Hsp 65 plasmid DNA was also
effective against a drug (isoniazid) resistant isolate of M.
tuberculosis. This therapeutic effect was associated with a switch from a type 2 to a
predominantly type 1 response, leading to CTLs that can be traced to antigen-specific
adjuvant effect of the plasmid DNA. Much of the adjuvant effect of DNA vaccines is traced
to unmethylated CpG motifs that induce IL-12 secretion by antigen presenting cells. In
this study, a plasmid coding for IL-12 was able to bring about the greatest reduction in
bacterial numbers in 11 weeks, although a 50:50 mixture of IL-12 and Hsp 65 plasmids led
to antagonism! In another experiment, DNA vaccine (Hsp 65) given in 3 injections towards
the end of chemotherapy (pyrazinamide + isoniazid) in infected mice completely
eliminated the residual bacteria, precluding the possibility of regrowth of residual
bacteria into drug-resistant forms, a real problem in developing countries. The authors
state in the note added in proof that the H sp 65 DNA therapy given 8 weeks after aerosol
infection of mice or gunea pigs indicate a substantial therapeutic benefit and this would
be very relevant to the human situation, since transmission of most human tuberculosis is
airborne. Besides, guneapig is a better model than mice, the latter exhibiting a
relatively higher innate resistance to tuberculosis. It would be a significant advance in
therapeutic strategy, if the DNA vaccine would decrease the dose and duration of drug
treatment in the case of patients with tuberculosis.
The first published reports from India indicate of
modest success in the development of DNA vaccines against rabies9 and Japanese
Encepahlitis Virus10 in experimental animals. Interestingly, the efficacy of
DNA vaccine (G protein) against rabies is correlated to levels of neutralizing antibodies,
whereas in the case of JEV (envelope protein), cell-mediated immunity appears to be the
major mechanism of protection.
There is a great expectation that DNA vaccines can
offer protection against dreadful infectious diseases in developing countries in view of
their temperature stability, besides being affordable for the poor when compared to
recombinant/cell culture vaccines. DNA vaccine would not need a cold chain for storage and
transportation, making it an ideal product for villages and remote areas. But, as a human
vaccine, genetic immunization has several hurdles to cross in view of the variable immune
responses observed as influenced by the nature of the encoded antigen, dosage, route of
administration, animal species, duration and type of immune response, that can be further
compounded in the outbred human population. The concern of chromosomal integration and
insertional inactivation by the administered naked DNA appears to have receded to the
background in view of results obtained from safety studies. Ultimately, DNA vaccine as
such or in combination with recombinant/cell culture vaccine or as an adjunct to
chemotherapy is most likely to become a tool to benefit mankind in the 21st century.
Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G.,
Jani, A. and Felgner, P. L., Science, 1990, 247, 14651468.
Ertl, H. C. J. and Xiang, Z. J., Immunol., 1996, 156,
Good, M. F., Kaslow, D. C. and Miller, L. H., Annu. Rev. Immunol.,
1998, 16, 5787.
Shi, Y. A. P., Hasnain, S. E., Sacci,
J. B., Holloway, B. P., Fujioka, H.,
Kumar, N., Wholhueter, R., Hoffman,
S. L., Collins, W. E. and Lal, A. A., Proc. Natl. Acad. Sci. USA, 1999, 96,
Wang, R., Doolan, D. L., Charoenvit, Y., Hedstrom, R. C., Gardner, M.
J., Hobart, P., Tine, J., Sedegah, M., Fellarme, V., Sacci, J. B. Jr., Kaur, M., Klinman,
D. M., Hoffman, S. L. and Weiss, W. R., Infect. Immun., 1998, 66, 41934202.
Hoffman, S. L., Rogers, W. O., Crucci, D. J. and Venter, J. C., Nat.
Med., 1998, 4, 13511353.
Calarota, S., Bratt, G., Nordlund, S., Hinkula, J., Leaandersson,
A.-C., Sandstorm, E. and Wahren, B., Lancet, 1998, 351, 13201325.
Lowrie, D. B., Tascon, R., Bonato, V. L. D., Lima, V. M. E.,
Faccioli, L. H., Stavoupoulos., Colstan, M. J., Hewinson, R. G., Moelling, K. and Silva,
C. L., Nature, 1999, 400, 269271.
Biswas, S., Ashok, M. S., Reddy, G. S., Srinivasan, V. A. and
Rangarajan, P. N., Curr. Sci., 1998, 76, 10121016.
Ashok, M. S. and Rangarajan, P. N., Vaccine, 1999, 18,
G. Padmanabhan is in the