Review Article

 

Vaccination: A Future Perspective

 

Shaswati Subhadarsini Pany1, Nihar Nalini Mohanty2, Laxmi Narayan Sarangi3*, Jajati Keshari Mohapatra4, Sathish B Shivachandra2

1Guru Angad Dev Veterinary & Animal Science University, Ludhiana, Punjab; 2Indian Veterinary Research Institute, Mukteswar; 3Regional Medical Research Centre, Bhubaneswar; 4Project Directorate on Foot-and-Mouth Disease, IVRI, Mukteswar, India.

Editor | Muhammad Munir (DVM, PhD), Avian Viral Diseases Program, Compton Laboratory, Newbury, Berkshire, RG20 7NN, United Kingdom.

Special Issue | 4(2015) “Advances in animal disease diagnosis, vaccines and therapeutics”

Received | December 27, 2014; Revised | January 23, 2015; Accepted | January 24, 2015; Published | February 21, 2015

*Correspondence | Laxmi Narayan Sarangi, Regional Medical Research Centre, Bhubaneswar, India; Email: [email protected]

Citation | Pany SS, Mohanty NN, Sarangi LN, Mohapatra JK, Shivachandra SB (2015). Vaccination: a future perspective. Adv. Anim. Vet. Sci. 3(4s): 1-8.

DOI | http://dx.doi.org/10.14737/journal.aavs/2015/3.4s.1.8

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2015 Pany et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

 

Introduction

 

Over the past years the field of vaccinology has undergone a substantial transformation (Flingai et al., 2013). Where earlier, civilisations were being wiped out due to unavailability of protection against certain diseases like plague, now it is being possible to eradicate diseases like small pox from the globe and control many dreaded diseases of human and veterinary significance (Kanampalliwar et al., 2013). There has been a shift in technology of production of vaccine candidate from physical and chemical inactivation of microorganisms to the production of recombinant microbes, deletion mutants and expression of putative proteins etc. The field of vaccinology is undergoing a rapid transformation by the gradual understanding and incorporation of recombinant DNA technology, molecular genetics, cellular immunology, structural biology, bioinformatics, computational biology, nanotechnology, formulation technologies and systems biology. More recently, new methods of antigen discovery and design as well as investigation of vaccine responses have been applied, including reverse vaccinology (Rinaudo et al., 2009).

 

While the development and widespread use of effective vaccines has an extraordinary impact on global health, there remain many infectious and other diseases for which vaccines are not available. In general, the viruses, bacteria, and parasites for which new vaccines are needed are either complex in their pathogenesis, exhibit extensive variability, or have evolved evasive mechanisms to bypass the immune system. The development of vaccines for many pathogens remains elusive, and there is a growing requirement for the fast development of effective vaccines for emerging diseases (Morens et al., 2008). Here, we discuss the technological advances that are propelling into a new era in vaccinology, highlighting the various aspects which remain the target of development, for combating the challenges of emerging infectious and non-infectious diseases. The cognition of gradual shift from the conventional vaccination strategy towards a more calculated and novel approach will not only keep human and animal sector abreast of the current trends in immunoprophylaxis but will also encourage research in a direction that will improve preparedness for coping up with the upcoming demands of the health sector.

 

A brief history

 

Vaccination surfaced as a result of cautiously planned attempt of humans in order to shield themselves from infectious diseases centuries ago when Indian Buddhist monks drank snake venom to confer immunity to snake bite. The Greek historian Thucydides in 429 BC observed that those who survived the plague in Athens did not become re-infected with the disease and from there the concept of immunity was born. In the eleventh century the Chinese insufflated variola scabs into nose to confer resistance against small pox, which was based on the observation of protection by prior exposure to the disease (Plotkin, 2005). Lady Mary Montague, wife of the English ambassador at Istanbul, introduced the practise of variolation in England. The common observation that milkmaids were generally immune to smallpox was the impulse for Jenner’s epochal hypothesis of prevention of small pox by exposure to cow pox, which was proved by inoculating the subject with pus in the blisters from cowpox of milkmaids and subsequent challenge with the virus. This discovery not only led to the eradication of small pox in the twentieth century, but also established the idea of developing immunity against an organism by prior exposure to it.

 

The illustration of ‘chance & prepared of mind’ could be more appropriately derived from Pasteur’s accidental discovery of attenuation in his own laboratory by ageing of a culture of fowl cholera. Based on this observation, Pasteur hypothesised the attenuation of pathogens by environmental and chemical insults and confirmed it by inventing vaccines for fowl cholera, anthrax and rabies (Plotkin, 2005). With the advent of time, the technique of attenuation by passaging organisms in artificial media and in unnatural host was a major breakthrough by Calmette and Guérin and Theiler, who used the methodology to attenuate Mycobacterium bovis and yellow fever virus respectively. The method of attenuating viral vaccines by propagating in cell culture scouted towards the development of several vaccines, including polio, measles, mumps, and rubella (Koff et al., 2013). Meanwhile, the concept of humeral and cellular basis of immunity was established, which provided a more precise approach towards regulation of immune response.

 

Conventional vaccination strategy

 

Pasteur’s early approaches to vaccine development, even today are the two yardsticks of vaccine technology. Existing vaccines can be divided into two broad groups: live and killed vaccines. Live vaccines induce immunity by mimicking natural infections using attenuated forms of pathogens while killed vaccines use the whole pathogen or their components to induce protective immunity. Conventional approach of vaccine production are mostly based on the cultivation of the microorganism in laboratory conditions followed isolation of individual components in pure form, which are finally tested for its immunogenicity with the exception to non-cultivable microorganisms. In many cases the predicted/assumed antigens expressed during natural infections fail to be expressed in laboratory conditions. Moreover, it has been observed that the proteins which are most abundant and easily purified may not provide adequate protection. (Capechi et al., 2004).

 

Reverse Vaccinology

 

The use of sequence data of microorganisms and bioinformatics tools for the design of vaccines is a recent approach towards antigen discovery and has been termed “Reverse vaccinology”. By taking into focus the whole genome sequences, current computer programs allow predicting the function or the putative cellular localization of the newly-identified open reading frames (ORFs) thus providing a virtual catalogue of all the candidate molecules which can be used as an effective vaccine. Moreover the genomic information is also used along with the amalgamation of “Functional Genomics” like in vivo expression technology (IVET), signature tagged mutagenesis (STM), DNA microarrays and proteomics (two-dimensional gel electrophoresis and mass spectrometry) in order to select novel antigens (Capechi et al., 2004). Besides reverse vaccinology, “antigenome analysis” using libraries of genetically expressed antigens and screening for immunogenicity of the proteins during infection, has also evolved (Giefing et al., 2008). Even the gradual evolution in mass spectrometry analysis has enabled direct quantification of bacterial surface antigens (Koff et al., 2013). Though, the aforementioned methods have enormous capability of identifying and tapping potential antigens for inclusion in vaccines, they remain limited in their capacity to predict antigens with protective ability. Efforts have also been made towards identification of subjects with broadly neutralizing antibody (bnAbs) serum responses, determination of the structure of the binding sites of bnAbs and mimicking the epitopic binding sites of such bnAbs by exploiting the memory cell repertoire of infected individual, for immunogens to elicit such bnAbs (Simek et al., 2009; Walker et al., 2009; Kwong et al., 2012; Burton et al., 2012; Zhou et al., 2010).

 

Proteomics in vaccine design

 

With the accessibility to genomic sequence data, advances in mass spectrometry analysis and an innumerable ways of characterizing proteins have made it possible to separate and identify proteins expressed in a cell. The entire set of proteins encoded by the genome has been defined as “proteome” (Grandi, 2001). In proteome analysis, a protein mixture is first resolved leading to the separation of individual components followed by proteolytic digestion with specific peptides to deliver discrete peptides. The peptides are evaluated by mass spectrometry and the experimental value is compared with all the predicted ones, thus ascertaining the product of a specific gene. (Capecchi et al., 2004). Analysis of the proteome allows identification of proteins under different growth conditions. This approach has been used extensively to identify novel vaccine candidates against several pathogens.

 

Adjuvants & modern delivery technologies

 

Adjuvants are molecules, potentiating the efficacy and longevity of specific immune response to antigens, with minimal toxicity and adverse effects (Wack et al., 2005). The addition of adjuvants to vaccines not only enhances the immune response, but also reduces the antigen concentration and dosage, effectively directing appropriate immune response. An effective adjuvant formulation utilizes multiple components and methodologies to achieve the desired immunological response (Schijns, 2000). Of the various mechanisms used, the most effective ones includes establishing antigen depots, enhancing antigen presentation by dendritic cells (DC) to effector cells like CD8+ cytotoxic T-lymphocyte (CTL) or CD4+ T-helper (Th) lymphocyte responses (Mohan et al., 2013). Adjuvants may also exert their activities by targeted distribution of antigen to specific cells, improving stability of antigen and by acting as immune modulators as in case of microbial and endogenous adjuvants (Dangi et al., 2011). Realizing the need of a powerful adjuvant, Freund in the mid-1930s, developed a powerful immunologic adjuvant known as Freund’s complete adjuvant (FCA) basically composed of a water-in-mineral oil emulsion containing killed mycobacteria. But today the adjuvant technology has transcended way beyond in search of a complete adjuvant, so as to revolutionize the modern era of vaccination. An adjuvant can be basically divided into two classes: delivery systems and immunopotentiators, mostly basing on their mechanism of action (Dangi et al., 2011). Immunostimulatory/ Immunopotentiating adjuvant act predominantly at the cytokine level or by activation of co-stimulatory signals, with due emphasis on the type of antigen used for optimal immune response (Mbawuike et al., 2007). However, on a different note, delivery systems emphasize on the presentation, protection and targeted delivery of the antigen. A brief description of some of the potential adjuvants which are/can be used in various vaccine formulations to achieve specific immune response through desired pathway has been described below.

 

Aluminium salts such as aluminium hydroxide, aluminium phosphate and potassium aluminium sulphate (alum) work by inducing inflammation and antigen retention at the injection site for a prolonged period thus allowing detection by the immune cells (Glenny et al., 1926). However, aluminium containing vaccines may be associated with local reactions. Liposomes on the other hand are vesicles in which an aqueous core is encapsulated within one or more phospholipid bilayers. Liposomes are highly flexible delivery systems, able to carry both hydrophobic and hydrophilic substances. They are relatively nontoxic and can be conjugated to antibodies or ligands and can be engineered for vaccine delivery, either by core or surface (Mohanty et al., 2014). Similarly, viral vectored vaccines are also delivery systems but differ from liposomes in the manner that viral-vectored vaccines consist of a non-replicating virus that contains genetic material from a pathogen to which specific immunity is desired. Such vectored vaccines stimulate both mucosal and systemic immunity and are commonly referred to as live recombinant vaccines (Plotkin, 2005). Adenovirus, which has been administered orally as its own vaccine for decades, has also provided a frequent vector platform for many of these types of vaccines, including delivery systems for tetanus (Shi et al., 2001) and influenza (Van Kampen et al., 2005; Vemula and Mittal, 2010) .

 

VLP (Virus like Particle) are essentially non-infective viruses with self-assembled viral envelope proteins without the accompanying genetic material. In this system the envelope of one virus is used as a framework to which additional components of the virus or another virus or pathogen are attached or inserted (Metcalfe et al., 2006). Both types of particles maintain morphology and cell penetrating ability similar to infective viral particles (Huckriede et al., 2005) and are capable of inciting both humoral and cell mediated immunity (Grgacic et al., 2006). Bacterial components are also being used in adjuvant formulations to enhance the immune response through various pathogen recognition receptors (PRR). Likewise Monophosphoryl lipid A is a TLR-4 receptor agonist (Persing et al., 2006) derived by detoxifying lipopolysaccharide (LPS) from Salmonella minnesota R595. After detoxification, the resulting MPL maintains adjuvacity and is a versatile vaccine adjuvant which in combination with other adjuvant systems can deliver more dynamic response (Baldridge et al., 1999). MPL has been shown to include cytokine release leading to activation of immune cells (Neuzil et al., 2006). Similarly CpG motifs are six deoxynucleotides-long DNA sequences with a central CpG dinucleotide, and are more frequent in bacterial DNA than that of mammalian DNA. CpG motifs are recognized by the Toll like receptor (TLR 9) (Cornelie et al., 2004) inducing the secretion of type I interferons and IL-12 by cells of the innate immune system, thus triggering Th1 cellular response. Therefore, CpG-containing DNA-based molecules have been advocated for therapeutic applications and also for adjuvanting other types of vaccines (Klinman, 2004).

 

Nowadays, a wide number of adjuvants are squalene based formulations. Squalene is a linear triterpene that is used mostly because of its stability and biocompatibility and is extensively utilized in parenteral emulsions for drug and vaccine delivery. Emulsions containing squalene facilitate solubilisation, enhanced release, and cell uptake of target molecules. Squalene and its hydrogenated form, squalane, both have been ideally suited for making stable and non-toxic emulsions. Combination adjuvant known as DETOX™ contains MPL® and Mycobacterium phlei cell wall skeletons in a squalene emulsion and has been used in the clinical case of melanoma (Mitchell et al., 1988) and ovarian cancer (MacLean et al., 1992). Such MPL®-containing vaccine formulations have been found to enhance both cellular and humoral immune responses with minimal toxicity (depending on the antigen) relative to non- MPL® formulations (Mitchell et al., 1988). A formulation known as MF59, which is an oil in water emulsion, squalene based vaccine adjuvant also significantly enhances immune response to a wide variety of antigens (Brito et al., 2011). However it has been observed to cause serious autoimmune disorders and hence seldom used in human vaccination except influenza vaccine. However in a more convenient approach Montanide™, including ISA 50V, 51, 206 and 720 which are either water-in-oil or oil-in-water emulsions are also used in strategic vaccine formulations. ISA 206 and 50V have been used only in veterinary vaccine formulations while the others are under investigation for use in humans (Aucouturie et al., 2002). The immune enhancement produced by the Montanide™ emulsions is believed to be due to the formation of a depot at the site of injection (Miles et al., 2005). These emulsions are under clinical trials for vaccines against malaria and various cancers (Miles et al., 2005; Toledo et al., 2001).

 

Routes of vaccination

 

Another vital aspect of vaccination is the route by which the formulations have to be delivered, without the proper channel the designed engineered vaccine formulation will not meet the targeted ends, irrespective of the generation to which it may belong. Traditional vaccine administration methodologies are limited by their nature of inducing local reaction, inconvenience, safety and cost effectiveness. Very few vaccines are being administered intranasal, orally and subcutaneously for diseases like influenza, polio, and measles-mumps-rubella respectively (Plotkin, 2005). Ongoing research on alternative experimental administration strategies includes ballistic delivery to skin (the gene gun), the transdermal patch and other intradermal methods, plus sublingual, aerosol, rectal and vaginal mucosal vaccines. The main advantages of alternative delivery strategies are the potential to induce immune responses at the common portals of pathogen entry (e. g. oral polio vaccine replicating in the gut), convenience (e. g. ease of use of the transdermal patch), combination of vaccines to reduce or simplify the vaccination schedule, and reduction or elimination of administration via standard hypodermic needle injection. Despite the intuitive value of these approaches, few vaccines today are administered via non-IM routes. This is for several reasons including feasibility, lack of proven efficacy and limited safety data. (Stanberry and Strugnell, 2011).

 

The inconvenience and apprehensions of a needle piercing the body is prevalent in both animal and human health care sector. The closest development in this area is the transdermal application (Matyas et al., 2004). Many devices have been developed to deliver antigens across the skin by means of patches infused with adjuvant applied to mild skin abrasion using micro-needles to pierce the stratum corneum. Once past the superficial layer, it enhances contact with dendritic cells, thus potentiating immune response (Plotkin, 2005).

 

A peek into future targets

 

Till date the concept of immunization used to encompass only infectious diseases but in near future goals are being set for targeting non infectious diseases like cancer, and desensitization methods of tackling allergies with better antigens for inducing IgG rather than IgE antibodies are in development (Linhart et al., 2012). It is also intriguing that individuals with inherited mutations that predispose to cancer might be immunized prophylactically before cancer develops. Attempts are also being made towards tolerance to auto antigens (Larsson et al., 2011), contraception by immunization against hormones (Vizcarra et al., 2012) combating multiple ailment by immunization against cholesterol (Fattori et al., 2012) and antibody mediated clearance of drugs (Kinsey, 2014). Moreover the use of mice models to predict and study the immune response has emerged with various setbacks of difference in dosage as compared to animals and humans and even the minor variations in inciting an immune response in mice will have significant impact in predicting the effect in higher targets. So efforts have been made towards development of humanized mice which are very promising in mimicking human immune response are being validated (Koff et al., 2013).

 

Conclusion

 

Despite the immense advancements made in the field of vaccinology there are many puzzles yet to be deciphered, also many challenges keep popping up as we go deep in search of solutions. Moreover the lack of understanding of host-pathogen interaction, immunological responses under varying conditions and the inability of fine tuning protective response with antigenic variability is irrefutable. The radical changes in this field has to be incorporated in both human and animal sectors irrespective of the targeted aetiology, as the basic fundamental methodology of formulation and devising of an effective vaccine strategy remains the same. The ongoing project against major global killers such as AIDS, tuberculosis, malaria, and other infectious diseases should revolutionise the health sector as a whole thus emphasizing more on successful vaccine development against allergies, autoimmune diseases, disease of zoonotic importance and cancers; and provide a novel vaccines against new and emerging diseases.

 

References

 

Aucouturier J, Dupuis L, Deville S,Ascarateil S, Ganne V (2002). Montanide ISA 720 and 51: a new generation of water in oil emulsions as adjuvants for human vaccines. Expert Rev Vaccines 1:111–118.

Baldridge JR, Crane RT (1999). Monophosphoryl lipid A (MPL) formulations for the next generation of vaccines. Methods. 19(1): 103-107. http://dx.doi.org/10.1006/meth.1999.0834

Brito LA, Chan M, Baudner B, Gallorini S, Santos G, O’Hagan DT, Singh M (2011). An alternative renewable source of squalene for use in emulsion adjuvants. Vaccine. 29(37): 6262-6268. http://dx.doi.org/10.1016/j.vaccine.2011.06.067

Burton DR, Poignard P, Stanfield RL, Wilson IA (2012). Broadly neutralizing antibodies present new prospects to counter highly antigenically diverse viruses. Science. 337(6091): 183-186. http://dx.doi.org/10.1126/science.1225416

Capecchi B, Serruto D, Adu- Bobie J, Rappuoli R, Pizza M (2004). The Genome Revolution in Vaccine Research. Curr. Issues Mol. Biol. 6: 17-28.

Cornelie S, Hoebeke J, Schacht AM, Bertin B, Vicogne J, Capron M (2004). Direct evidence that toll-like receptor 9 (TLR9) functionally binds plasmid DNA by specific cytosine-phosphate-guanine motif recognition. J. Biol. Chem. 279: 15124-15129. http://dx.doi.org/10.1074/jbc.M313406200

Dangi AA, Sheth NR, Sodha HH, Joshi PC, Bhalodiya DS, Panchal AC, Ramanuj PR (2011). Formulation and development of vaccines and their selection for next generation. Bull. Pharm. Res. 1(3): 49-62.

Fattori E, Cappelletti M, Surdo PL, Calzetta A, Bendtsen C, Ni YG, Monaci P (2012). Immunization against proprotein convertase subtilisin-like/kexin type 9 lowers plasma LDL-cholesterol levels in mice. J. Lipid Res. 53(8). 1654-1661. http://dx.doi.org/10.1194/jlr.M028340

Flingai S, Czerwonko M, Goodman J, Kudchodkar SB, Muthumani K, Weiner DB (2013). Synthetic DNA vaccines: improved vaccine potency by electroporation and co-delivered genetic adjuvants. Front. Immunol. 4:354. http://dx.doi.org/10.3389/fimmu.2013.00354

Giefing C, Meinke AL, Hanner M (2008). Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies. J. Exp. Med. 205(1): 117-131. http://dx.doi.org/10.1084/jem.20071168

Glenny AT, Pope CG, Waddington H, Wallace U (1926). Immunological Notes: XVII–XXIV. J. Pathol. Bacteriol. 29: 31-40. http://dx.doi.org/10.1002/path.1700290106

Glück R, Moser C, Metcalfe IC (2004). Influenza virosomes as an efficient system for adjuvanted vaccine delivery. Expert Opin. Biol. Ther.  4(7): 1139-1145. http://dx.doi.org/10.1517/14712598.4.7.1139

Grandi G (2001). Antibacterial vaccine design using genomics and proteomics. Trends Biotechnol. 19: 181-188. http://dx.doi.org/10.1016/S0167-7799(01)01600-6

Grgacic EV, Anderson DA (2006). Virus-like particles: passport to immune recognition. Methods. 40(1): 60-65. http://dx.doi.org/10.1016/j.ymeth.2006.07.018

Huckriede A, Bungener L, Stegmann T, Daemen T, Medema J, Palache AM, Wilschut J (2005). The virosome concept for influenza vaccines. Vaccine. 23: S26-38. http://dx.doi.org/10.1016/j.vaccine.2005.04.026

Kanampalliwar AM, Soni R, Girdhar A, Tiwari A (2013). Reverse Vaccinology: Basics and Applications. J. Vaccin. 4: 194.

Kinsey B (2014). Vaccines against drugs of abuse: where are we now? Ther. Adv. Vaccines. 2(4): 106-117. http://dx.doi.org/10.1177/2051013614537818

Klinman DM (2004). Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat. Rev. Immunol. 4(4): 249-258. http://dx.doi.org/10.1038/nri1329

Koff WC, Burton DR, Johnson PR, Walker BD, King CR, Nabel GJ, Ahmed R, Bhan MK, Plotkin SA (2013). Accelerating Next-Generation Vaccine Development for Global Disease Prevention. Science. 340: 1232910. http://dx.doi.org/10.1126/science.1232910

Kwong PD, Mascola JR (2012). Human antibodies that neutralize HIV-1: identification, structures, and B cell ontogenies. Immunity. 37(3): 412-425. http://dx.doi.org/10.1016/j.immuni.2012.08.012

Larsson HE, Lernmark A (2011). Vaccination against type 1 diabetes. J. Intern. Med. 269(6): 626-635. http://dx.doi.org/10.1111/j.1365-2796.2011.02386.x

Linhart B, Valenta R (2012). Mechanisms underlying allergy vaccination with recombinant hypoallergenic allergen derivatives. Vaccine. 30(29): 4328-4335. http://dx.doi.org/10.1016/j.vaccine.2011.11.011

MacLean GD, Bowen-Yacyshyn MB, Samuel J, Meikle A, Stuart G, Nation J, Poppema S, Jerry M, Koganty R, Wong T (1992). Active immunization of human ovarian cancer patients against a common carcinoma (Thomsen-Friedenreich) determinant using a synthetic carbohydrate antigen. J. Immunother. 11(4): 292-305. http://dx.doi.org/10.1097/00002371-199205000-00008

Matyas GR, Friedlander AM, Glenn GM, Little S, Yu J, Alving CR (2004). Needle-free skin patch vaccination method for anthrax. Infect. Immun. 72: 1181-1183. http://dx.doi.org/10.1128/IAI.72.2.1181-1183.2004

Mbawuike I, Zang Y, Couch RB (2007). Humoral and cell-mediated immune responses of humans to inactivated influenza vaccine with or without QS21 adjuvant. Vaccine. 25(17): 3263-3269. http://dx.doi.org/10.1016/j.vaccine.2007.01.073

Metcalfe IC, Glück R (2006). Virosomes for vaccine delivery, Schijns, V.E.J.C & O’Hagan, D.T. (Eds.), Immunopotentiators in Modern Vaccines.179-189.

Miles AP, McClellan HA, Rausch KM, Zhu D, Whitmore MD, Singh S, Martin LB, Wu Y, Giersing BK, Stowers AW, Long CA, Saul A (2005). Montanide ISA 720 vaccines: quality control of emulsions, stability of formulated antigens, and comparative immunogenicity of vaccine formulations. Vaccine. 23: 2530-2539. http://dx.doi.org/10.1016/j.vaccine.2004.08.049

Mitchell MS, Kan-Mitchell J, Kempf RA, Harel W, Shau HY, Lind S (1988). Active specific immunotherapy for melanoma: phase I trial of allogeneic lysates and a novel adjuvant. Cancer Res. 48(20): 5883-5893.

Mohan T, Verma P, Rao DN (2013). Novel adjuvants & delivery vehicles for vaccines development: A road ahead.  Indian J. Med. Res. 138(5): 779-795.

Mohanty NN, Palai TK, Prusty BK, Mohapatra JK (2014). Nanomedicine in Veterinary Science. Vet. Res. Int. 2(4): 90-95.

Morens DM, Folkers GK, Fauci AS (2008). Emerging infections: A perpetual challenge. Lancet Infect. Dis. 8: 710-719. http://dx.doi.org/10.3201/eid1409080952

Neuzil KM, Jackson LA, Nelson J, Klimov A, Cox N, Bridges CB, Dunn J, DeStefano F, Shay D (2006). Immunogenicity and reactogenicity of 1 versus 2 doses of trivalent inactivated influenza vaccine in vaccine-naive 5-8-year-old children. J. Infect. Dis. 194(8): 1032-1039. http://dx.doi.org/10.1086/507309

Persing DH, McGowan P, Evans JT, Cluff C, Mossman S, Johnson D, Baldridge JR (2006). Toll-like receptor 4 agonists as vaccine adjuvants, V.E.J.C. Schijns, D.T. O’Hagan (Eds.), Immunopotentiators in Modern Vaccines, Academic Press, Burlington, MA. 93-107.

Plotkin SA (2005). Vaccines: past, present and future. Nature Medicine. 11: S5-S11. http://dx.doi.org/10.1038/nm1209

Rinaudo CD, Telford JL, Rappuoli R, Seib KL (2009). Vaccinology in the genome era. J. Clin. Invest. 119: 2515-2525. http://dx.doi.org/10.1172/JCI38330

Schijns VE (2000). Immunological concepts of vaccine adjuvant activity. Curr. Opin. Immunol. 12: 456-463. http://dx.doi.org/10.1016/S0952-7915(00)00120-5

Simek MD, Rida W, Priddy FH, Pung P, Carrow E, Laufer DS, Lehrman JK, Boaz M, Tarragona-Fiol T,Miiro G, Birungi J, Pozniak A, McPhee DA, Manigart O, Karita E, Inwoley A, Jaoko W, Dehovitz J, Bekker LG, Pitisuttithum P, Paris R, Walker LM, Poignard P, Wrin T, Fast PE, Burton DR, Koff WC (2009). Human immunodeficiency virus type 1 elite neutralizers: individuals with broad and potent neutralizing activity identified by using a high-throughput neutralization assay together with an analytical selection algorithm. J. Virol. 83(14): 7337-7348. http://dx.doi.org/10.1128/JVI.00110-09

Shi Z, Zeng M, Yang G, Siegel F, Cain LJ, van Kampen KR, Elmets CA, Tang DC (2001). Protection against tetanus by needle-free inoculation of adenovirus-vectored nasal and epicutaneous vaccines. J. Virol. 75(23): 11474-11482. http://dx.doi.org/10.1128/JVI.75.23.11474-11482.2001

Stanberry LR, Strugnell R (2011). Vaccines of the future: Understanding Modern Vaccines. Perspect. Vaccinol. 1(1): 151-199. http://dx.doi.org/10.1016/j.pervac.2011.05.006

Toledo H, Baly A, Castro O, Resik S, Laferté J, Rolo F, Navea L, Lobaina L, Cruz O, Míguez J, Serrano T, Sierra B, Pérez L, Ricardo ME, Dubed M, Lubián AL,Blanco M, Millán JC, Ortega A, Iglesias E, Pentón E, Martín Z, Pérez J, Díaz M, Duarte CA (2001). A phase I clinical trial of a multi-epitope polypeptide TAB9 combined with Montanide ISA 720 adjuvant in non-HIV-1 infected human volunteers. Vaccine. 19(30): 4328-4336. http://dx.doi.org/10.1016/S0264-410X(01)00111-6

Wack A, Rappuoli R (2005). Vaccinology at the beginning of the 21st century. Curr. Opin. Immunol. 17: 411-418. http://dx.doi.org/10.1016/j.coi.2005.05.005

Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, Goss JL, Wrin T, Simek MD, Fling S, Mitcham JL, Lehrman JK, Priddy FH, Olsen OA, Frey SM, Hammond PW, Kaminsky S, Zamb T, Moyle M, Koff WC, Poignard P, Burton DR (2009). Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science. 326(5950): 285-289. http://dx.doi.org/10.1126/science.1178746

Van Kampen KR, Shi Z, Gao P, Zhang J, Foster KW, Chen DT, Marks D, Elmets CA, Tang DC (2005). Safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans. Vaccine. 23(8): 1029-1036. http://dx.doi.org/10.1016/j.vaccine.2004.07.043

Vemula SV, Mittal SK (2010). Production of adenovirus vectors and their use as a delivery system for influenza vaccines.  Expert. Opin. Biol. Ther. 10(10): 1469-1487. http://dx.doi.org/10.1517/14712598.2010.519332

Vizcarra JA, Karges SL, Wettemann RP (2012). Immunization of beef heifers against gonadotropin-releasing hormone prevents luteal activity and pregnancy: Effect of conjugation to different proteins and effectiveness of adjuvants. J. Anim. Sci. 90(5): 1479-1488. http://dx.doi.org/10.2527/jas.2011-4554

Zhou T, Georgiev I, Wu X, Yang ZY, Dai K, Finzi A, Kwon YD, Scheid JF, Shi W, Xu L, Yang Y, Zhu J, Nussenzweig MC, Sodroski J, Shapiro L, Nabel GJ, Mascola JR, Kwong PD (2010). Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science. 329(5993): 811-817. http://dx.doi.org/10.1126/science.1192819