MAINTAINING AND ENHANCING VACCINE IMMUNOGENICITY

MAINTAINING AND ENHANCING VACCINE IMMUNOGENICITY

NEW VACCINES AND NEW VACCINE TECHNOLOGY 0891-5520/99 $8.00 + .OO MAINTAINING AND ENHANCING VACCINE IMMUNOGENICITY Jeremy D. Gradon, MD, and Larry I...

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NEW VACCINES AND NEW VACCINE TECHNOLOGY

0891-5520/99 $8.00

+ .OO

MAINTAINING AND ENHANCING VACCINE IMMUNOGENICITY Jeremy D. Gradon, MD, and Larry I. Luhvick, MD

This article highlights factors involved with maintaining and enhancing antigen delivery or immunogenicity. The article is meant as an overview of the field and, limited by size constraints, cannot do justice to any one of the areas discussed. The technological understanding, however, that underlies these advances, no doubt, is about to revolutionize vaccinology, in the near future. PRESERVATION OF THE COLD CHAIN

Initially, the term cold chain was coined to describe a method for distributing food that might otherwise spoil if not kept under refrigerated conditions. The concept was applied to the handling of vaccines in the early 1970~.'~* Upon introduction, it became immediately apparent that close cooperation and coordination of many factors was required to achieve reproducible, successful distribution of potent vaccine from the manufacturer to often harsh field conditions. Major issues included among these considerations are as follows: Suitable stabilization Appropriate packing Temperature control

From the Division of Infectious Diseases, Department of Medicine, Sinai Hospital; and The Johns Hopkins School of Medicine, Baltimore, Maryland OX); and the Division of Infectious Diseases, Department of Medicine, VA Medical Center, Brooklyn; Department of Veteran Affairs, New York Harbor Health Care System; and the SUNY-Health Science Center at Brooklyn, Brooklyn, New York (LIL) ~~

~

INFECTIOUS DISEASE CLINICS OF NORTH AMERICA VOLUME 13 * NUMBER 1 MARCH 1999

39

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GRADON & LUTWICK

Timely delivery from manufacturer to user Timely use, reconstitution, and administration Education of involved personnel of the fragility of the vaccine and need to maintain appropriate conditions for shipping and storage Disaster management plan for events, such as refrigerator power loss Continuous quality control monitoring This section deals primarily with the maintenance of vaccines at appropriate temperatures. Examples of data pertaining to the level of knowledge among practicing physicians of the need to adhere to cold chain requirements are also briefly reviewed. It should be noted that the same issues exist for veterinary vaccines as well.93 Failure to protect vaccines from exaggerated cold conditions as well as excess heat can limit product viability. With this in mind, Table 1 describes the ideal temperatures at which some common vaccines must be kept. Although it is intuitive to assume that the major problems to be encountered in the integrity of cold chain maintenance are in the field of the developing world, there are strong data to suggest that physicians practicing in North America, Europe, and Australia have a poor knowledge base and poor training regarding this important This is highlighted by a study from Los Angeles that demonstrated that in 50 pediatric practices, 84% of vaccine coordinators were unaware of appropriate storage temperatures for vaccines and 36% were unaware that freezing inactivated many vaccine^.^ In the developing world, the problems faced are a combination of lack of knowledge, resources, and infrastructure together with more extreme environmental conditions. Steps taken by the World Health Organi~ation~~ and other bodies to assist in ensuring that the integrity of cold chain is maintained include the following: Routine use of the cold chain monitor, a device that detects exposure of a vaccine lot to temperature higher than 10°C. Activation is achieved by peeling off a plastic strip. If the monitor is then exposed to temperature higher than 12"C, window A turns color. Window B turns color if exposed to 12°C for 8 days or 21°C for 6 days, and window C does the same if ambient temperature around the monitor is 12°C for 14 days or 21°C for 11 days. Exposure to temperature higher than 34°C turns window D a color within 2 hours. Use of the freeze watch monitor. This is a device that monitors if the vaccine lots are inadvertently frozen to lower than -4°C for more than 1 hour. Use of ice-limed refrigerators for the developing world. These refrigerators are able to function on 8 hours of electrical supply per day, maintaining cold temperatures by forming an internal layer of ice. Avoid the use of kerosene powered refrigerators. These products have been shown to be unreliable, even when well-trained staff

~~~~

~~~~

~~

Unstable Stable if frozen solid ( - 11°C to - 18°C) Stable when frozen (not diluent) Stable for -2 y

Stable for 18-24 mo Stable 3-12 mo

Stable for -18 mo

Stable for -2-3 y

Stable and potent for -30 d

Stable and potent for -2 Y Stable for -12 rno

Monovalent stable for 3 d. Some DFT products stable for -4 mo Stable for -4 mo Unstable, 50% loss in potency in 20 d Stable for -1 mo

Variable between strains but is likely to lose potency over 3 mo Stable for 6 mo

22-25°C

24°C

*Fmm Guthridge SL, Miller N C Cold chain in a hot climate. Aust N 2 J Public Health 20657, 1996;with permission.

~~~~~~~

Measles-mumps-rubella (freeze dried; reconstitution will produce rapid instability at room temperature)

Diphtherialtetanus/pertussis Oral polio vaccine

Pertussis

Stable if frozen

Bacille Calmette-Gu6rin (freeze dried; reconstituted it becomes unstable rapidly at room temperature) Diphtheria/tetanus toxoids Tetanus is instable with freezing Significant loss of potency if frozen 4 - 5°C

Unstable

Below 0°C

Hepatitis B

Vaccine

Table 1. VACCINE STABILITIES AT FREEZING, OPTIMUM, AND AMBIENT TEMPERATURES

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GRADON & LUTWICK

with adequate supplies of fuel and spare parts are available, in attempting to store vaccines adequately. Avoid storing vaccines in the door compartments of domestic refrigerators where it is more difficult to maintain the correct temperature. Authorities acknowledge that the ban of such refrigerators is unrealistic. Use of a "vaccine only'' dedicated refrigerator. Monitoring practices have seemingly made a difference in maintaining vaccine potency as shown by assaying potency. In one report,19 an Indian state with a program for cold chain maintenance had 92.8% of trivalent poliovirus vaccine samples retaining potency, whereas only 72% were found potent in a state without such a program. Examples of problems encountered with these practices are highlighted in recent studies from Australia, Ireland, Sri Lanka, and Colorado. Guthridge and MillerN describe the problems encountered in the distribution of vaccines throughout Australia following the release of the vaccines from one of the recognized national vaccine distributors. Temperature sensors monitored vaccine conditions for a total of 8369 hours. One lot of vaccine was found to have been exposed to temperatures below freezing for 405 hours. A second lot was maintained at acceptable conditions until it was placed in Styrofoam containers for transport to rural health centers, at which time temperatures rose above acceptable limits. A third lot did well until delivery to a rural general practitioner's office, where the vaccine was inadvertently frozen for 14 hours overnight. Thus, despite the high ambient temperatures in Australia overall, the bulk of clinical problems with the cold chain related to excessive cooling rather than warming of vaccines. Another Australian study echoed this, finding 21 of 25 electronically monitored vaccine storage sites had adverse exposures to unacceptable cold It should be noted that these results were obtained after both a 1990 report that had highlighted similar problems and multiple interventions to maintain the cold chain in Australia had been implemented. Finnegan and Howellz6reviewed the practices of 142 general practitioners in Ireland. They, as well, found that multiple interruptions in the maintenance of the cold chain occurred. Of note, 22% of vaccine lots were shipped to the general practitioner by standard mail without refrigeration at all. Additionally, only 8% of refrigerators had a thermometer in place, of which only two were looked at regularly and only 62% of the refrigerators stayed in the recommended range of +2"C to +8"C. Finally, it appeared to be a common practice to allow vaccines to stay at ambient temperature during routine refrigerator defrosting. Senanayake et a17*describe the effects of electricity power cuts on vaccine storage in Sri Lanka. The study was carried out during a period of time in which daily 2-hour power cuts were enforced. No vaccination center had alternative means of refrigeration and none had their own independent power generators. Impaired potency of both measles and diphtheria-tetanus-pertussisvaccines were found. When the study was

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repeated during a time of continuous electricity supply, full vaccine potency was found to be maintained. Salts, such as magnesium chloride, have been utilized to minimize losses related to inability to maintain temperatures in the refrigerator range. Such stabilizers have had some impact on the use of vaccines, such as measles and poliovirus, in tropical areas. It has also been shown that the fall in immunogenicity, in part related to hydrolysis of viral genomic RNA by intrinsic and extrinsic digestion by RNase, can be greatly inhibited by reducing pH below 7, which suppresses this enzymatic That a lack of compliance with appropriate vaccine storage temperatures is a problem in the United States as well is without question. As an example, a survey of 27 vaccine-use sites in Colorado9*found that only two had refrigerator temperatures that remained in an acceptable range: 63% fell below; 59% above; and 93% above, below, or both. No correlation was found between appropriate temperatures and either the presence of a designated cold chain monitor or the presence of a permanent refrigerator thermometer. It can thus be seen that the education of personnel who handle vaccines remains a vital component in the maintenance of cold chain integrity. The experience from Sri Lanka highlights that even when the staff are proficient, external factors beyond the control of the health care provider can interrupt the chain. Overall, for the purposes of the physician practicing in North America, it is important that vaccines be kept in (but not on the door of) dedicated refrigerators and that refrigerator temperature be regularly checked and monitored. Any vaccine that is delivered in a non-temperature-controlled fashion or if temperature monitors are not included or have been triggered by temperature variations should not be used and returned to the distribution site. The National Immunization Program of the Centers for Disease Control and Prevention has validated a number of approaches to be used in the packing and shipment of vaccines.41These procedures are outlined next:41 Frozen Vaccines Well-insulated containers reduce evaporation of dry ice Material used should enable packing to maintain cold chain over a full range of environmental conditions Freeze dried measles vaccine may be shipped this way but diluent should not be frozen Refrigerated Vaccines Frozen cold packs should always be used to pack vaccines for shipment in hot climates and may be used in warm weather Cold packs taken directly from the freezer should be exposed to ambient temperature for 15 to 30 minutes so their temperature rises to -5°C before use. This helps to avoid damaging vaccines that are unstable with freezing Nonfrozen cold packs at an initial temperature of approximately 5°C should cover at least four and preferably all six faces of the well-insulated box and protect vaccines not only in warm,

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temperate conditions but also from freezing in very cold conditions. Timely transportation is necessary to deliver the vaccine even in a well-packed and adequately insulated box. The costs of materials for reliable protection should be in most cases less than 1%of the cost of the vaccines. The cost of shipping replacements and personnel costs when delivery delays occur can be avoided by adequate packaging. ADJUVANTS: INCREASING IMMUNOGENICITY

Antigens used as vaccines vary greatly in their innate immunogenicity. Factors involved in determining the immunogenicity of an antigen include the physical properties of the antigen itself (e.g., polysaccharide or protein); dose and route of antigen delivered; the major histocompatibility complex (MHC) of the host56; and the nutritional and immune state of the host.14 In an attempt to enhance the immunogenicity of vaccines, adjuvants are added to less immunogenic, nonreplicative vaccines. In general, small peptides and purified polysaccharides are poorly immunogenic and their clinical usefulness is efianced either by fusing them with an immunogenic protein, such as tetanus toxoid (conjugation), or by the addition of an adjuvant. The use of adjuvants to enhance vaccine immunogenicity was intro.~~ a vast array of substances have been duced in the 1 9 2 0 ~ Although evaluated for use as adjuvants, to date only aluminum-containing compounds, such as alum, aluminum hydroxide, and aluminum phosphate, are approved for use in h~mans.6~ Recent work has suggested that the commercial adjuvant aluminum hydroxide is a poorly crystalline aluminum oxyhydroxide with the structure of the mineral boehmite and both alum and aluminum phosphate are amorphous aluminum hydro~yphosphate.~~ These compounds have a high surface area and a high protein adsorptive capacity for positively charged proteins. Analysis of these compounds could provide a framework for assessing the complex interactions between antigen and adjuvant to optimize the stability and efficacy of the product. The precise mechanisms whereby adjuvants enhance vaccine efficacy are generally unknown; however, the proposed modes of adjuvant activity have been recently extensively reviewed by Cox and Coulterlb and include the following: Irnmunomodulation: Here a substance is given separately from the actual vaccine, which causes a stimulation of the cytokine network, enhancing the host response to the vaccine. It has been that certain adjuvants stimulate a humoral response, whereas others increase the cytokines associated with a cellulartype response. Therefore, proper adjuvation can select the most appropriate response for a given pathogen. Indeed, the cytokines themselves can be used as vaccine adjuvants to direct the immune

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response in the correct manner.44Studies with recombinant bovine interleukin-1 in cattle have suggested that such an adjuvant might be best for diseases where protection is mediated by high antibody le~els.4~ Presentation: More effective immune responses can be facilitated by improving the presentation of the antigen by antigen-presenting cells to the immune system. Such adjuvants help B cells present antigen more effectively to T cells in combination with class I1 MHC. Enhancement of a cytotoxic T-lymphocyte clone: This may be accomplished via a class I MHC mechanism. Targeting: This can allow more efficient delivery of the immunogen to the appropriate cells by such mechanisms as improved delivery to macrophages or other cells through which the subsequent immune response can be augmented. The use of antigencontaining microparticles is an example of targeting. Production of oxygen radicals: Di Gianni et alZ1have observed that a variety of antioxidants were able to inhibit the enhancement of oil-based adjuvants on sheep blood cell immunogenicity. These antioxidants (catalase, N-acetylcysteine, glutathione) did not affect the antigen’s basal immune response. This observation suggests that oxygen radicals can have a crucial effect in at least some adjuvant effects. Production of a long-term, slow-release depot: This mechanism allows for antigen to remain trapped at the site of injection and not cleared from the body as readily, providing sustained stimulation of an immune response. Overall, it is possible to classify adjuvants into two main classes: (1) particulate and (2) nonparticulate. Particulate adjuvants exist as small particles, providing maximum immunologic benefit when the antigen is incorporated into or attached to the particle. In contrast, the nonparticulate adjuvant’s activity is not related to their physical nature but rather to immunomodulation or improved antigen targeting. The names and properties of some particulate and nonparticulate adjuvants are shown in Tables 2 and 3. When the decision to add an adjuvant to a vaccine is being made, theoretical considerations regarding the kind of immune response promoted by a particular adjuvant come into play regarding the potential adjuvant ~ h o i c eFor . ~ example, if a humoral response is felt to be protective optimally, the aluminum-containing adjuvants are indicated. These substances primarily stimulate a Th2 response inducing humoral immunity.” If cellular immunity is felt to be needed primarily (e.g., for an intracellular pathogen), then a Thl stimulating substance, such as QS21 (a derivative of the cholesterol-saponin mixture Quil l), may be better when available. Another moiety with prominent effects as an adjuvant in stimulating a Thl or cellular immune response is granulocyte-macrophage col-

~~

+

-

-

+ ++ + ++ ++++ + ++

-

+

Targeting

~

+++

-

-

+++

-

-

++++ ++

-

Presentation

~

~

-

-

-

++++ ++

-

-

-

CTL Induction

classification and review of their modes of action. Vaccine 15248 1997, with permission.

Moderate Thl & Th2 Moderate Thl Moderate Thl & Th2

-

-

-

Strong Th2 Weak Thl & Th2 Weak Thl & Th2 Strong Thl & Th2

lmmunomodulation

From Cox JC, Coulter AR: Adjuvants-a

Aluminum salts Water-oil emulsion Oil-water emulsion ISCOMs Liposomes 4 0 pm microparticles >10 pm microparticles Calcium salts Virosomes Stearyl tyrosine Gamma-inulin A1gammulin

Adjuvant

Table 2. CHARACTERISTICS OF PARTICULATE ADJUVANTS

+ + + (long term) + (short term) + (short term) + (short term)

-

-

+ (short term) + + + (short term)

Depot

-

Strong Thl

Various

Moderate Thl

Lipid A

Cytokines

Carbohydrate polymers Derivatized polysaccharides

-

-

-

+++ +++

-

-

+

-

-

-

CTL Induction

-

+++

-

Presentation

From Cox JC, Coulter AR: Adjuvants-a classification and review of their modes of action. Vaccine 15248, 1997, with permission

?

-

Strong Thl & Th2

Saponins

-/+

-

-

Targeting

?

Strong Th2. Strong Thl

lrnmunomodulation

Muramyl-dipeptide, hydrophilic Muramyl-dipeptide, lipophilic Nonionic block copolymers

Adjuvant

Table 3. CHARACTERISTICS OF PRINCIPAL NONPARTICULATE ADJUVANTS

?

Use in water-oil emulsion Use in oil-water emulsion Either oil-water or wateroil Form ISCOMs, use with liposomes Use with oil-water, liposomes, saponins Use with particulate adjuvants Conjugate to antigen

Comments

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GRADON & LUTWICK

ony-stimulating factor (GM-CSF). Indeed, GM-CSF, which stimulates the growth of antigen-presenting cells, such as dendritic cells (DC) and macrophages, appears to be quite a potent adjuvant for the generation of immune responses, both antibody and cellular in quality.22Additionally, oil-in-water emulsions containing one or more bacterial-derived immunostimulants (monophosphoryl lipid A [MPL] preparation; synthetic trehalose dicorynomycolate or Mycobacterium phZei cell wall skeleton) were found to be equivalent in adjuvant properties in mice to Freund’s complete adjuvant? Somewhat surprisingly, another potential adjuvant is DNA. Synthetic oligodeoxynucleotides containing unmethylated CpG motifs can potentiate clinical expansion of T cells responding to a specific peptide not coded for by the DNA.83 Mucosal administration of any vaccine can improve patient compliance by allowing immunization without the use of a needle. Generically, detoxified toxins, such as heat-labile Escherichiu coZi enterotoxin (LT) and the closely related cholera toxin, can provide mucosal adjuvant function. The native compounds have not been well tolerated by humans. A variety of toxin-like molecules produced by site-directed mutagenesis have been developed that are active as mucosal adjuvants but are nontoxic. As reviewed by O’Hagan,’j7one of these molecules, LTK63, with a single amino acid substitution in the enzymatically active LT subunit, has significant potential. As an example, LTK63 was very effective in the potentiation of the immune response to a measles antigen when the two were given intranasally.7l A genetically engineered nontoxic cholera toxin also appears to have good potential in targeting immunomodulation.’ Mucosal immunization can also be facilitated by the use of antigens adsorbed to or incorporated in very small particles, such as nanoparticles, which can deliver the antigen directly to the local antigenprocessing cells. These particles are discussed later, functioning not only as an adjuvant but also as a novel delivery system. If both cellular and humoral immunity are required, an acceptable response may be induced by either immune-stimulating complexes (ISCOMs) or by MPL, a bacterial antigen. The ISCOM is an approximately 40-nm cagelike particulate adjuvant that is produced combining the saponin Quil A, phospholipid, and cholesterol. During assembly, both hydrophobic and amphipathic antigens can be incorporated into the ISCOM.47By optimizing the ratio of components in an ISCOM, an immune response of both higher magnitude and quality can be stimulated.8 Such tailor-made adjuvant strategies are not clinically available (only the aluminum-containing adjuvants are at present), although it is anticipated that they may become available and more commonplace in the near future once the relatively high incidence of adverse effects with the early candidate adjuvants are controlled. NEW VECTORS FOR ANTIGEN DELIVERY

The success of live, attenuated vaccines, such as measles, rubella, varicella, and the SaZmoneZla typhi Ty2la, in the prevention of disease has

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spawned a significant degree of investigation into immunization using recombinant, attenuated bacteria and viruses as immunogens. S. typhimurium Mutants. Because the genetics of S. typhimurium have been very intensely studied, a variety of attentuated strains have been produced for potential use as recombinant vaccines. Among the metabolic areas investigated to attenuate the bacillus are the synthetic pathways for aromatic amino acids, purines, and lipopolysaccharides as well as for cyclic adenosine monophosphate regulation. Heterologous genes can be introduced into the bacterium on plasmids or via chromosomal integration. The latter offers a higher level of genetic stability. Insertion should not interfere with adequate viability and growth and, ideally, can be into the bacterial gene to be attenuated. The recombinant antigens produced usually accumulate in the cytoplasm intracellularly. Slow leakage extracellularly may occur or release may come only with cell lysis. It is not clear that excretion from the cell or expression on cell surface are prerequisites for utility. Recently, an epitope delivery system using the Salmonella type I11 secretion system has been utilized to deliver antigens better.76 A host of foreign genes have been produced in this model from enteric bacteria (i.e., E . coli, Vibriu cholerae, and ShigeZla); tetanus toxoid fragments; herpes simplex; dengue, and hepatitis B viral antigens; as well as potential immunogens from Plasmodium fakiparum, Leishmania major, Echinococcus multilocularis, and Entamoeba hist~lytica.~~ Unfortunately, although immunogenic in animal models, some studies have shown minimal to no protection.18,46, 51* 91 As reviewed,I7 phase I and I1 trials with several Salmonella recombinants have been halted due to suboptimal immune responses or poor protective efficacy. Recombinant S. typhimurium strains, however, have been protective in other systems.l5, w74Attention to the type of attenuation in the vector may be necessary to elicit a protective effect. Some mutants are more immunogenic and protective in mice than the prototypical aromatic amino acid m ~ t a n t . 9 ~ Besides the vector attenuation itself, prior exposure to the vector strain can blunt responsiveness and if boosters are needed, a different serotype vector should be used? Listeria Monocytogenes. Because of unique properties, this grampositive bacillus may be an excellent target for this technology if it can be adequately attenuated. Listeria is taken up by host cell phagosomes and then escapes into the cell's cytoplasm via the effect of listeriolysin 0 on the vacuolar membrane.30,92 Additionally, proteins that Listeria monocytogenes secretes are targeted to both MHC class 1 and I1 pathways for antigen presentation. A strong cellular immune response is then induced. Viruses and fungi, among others, that are cleared by cellular immunity may be idea1 targets for this system. Evidence in animals now exists that this technology is effective. As examples, recombinant Listeria expressing lymphocytic choriomeningitis virus antigen has shown protection in mice against subsequent viral challenge79and strains producing influenza virus nucleoprotein were effective in provoking more rapid clearance of influenza virus in mice.39

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GRADON & LUTWICK

Furthermore, a role may be played for recombinant Listeria in cancer immunology by inducing tumor-specific immunity resulting in antigenspecific rejection and regression of pre-existing tumors.70 Bacille Calmette-GuCrin. There are a number of reasons why bacille Calmette-GuQin (BCG) could be a unique vehicle for foreign gene products? it is the most widely used vaccine globally, it can be given at or any time postnatally, it is unaffected by maternal antibodies, it can sensitize for as long as 50 years, it is very heat stable, and it is not expensive to produce. Additionally, although it is used intradermally, BCG can and was initially given orally.82Although these characteristics suggest that recombinant BCG could be a major factor in attuenuated, recombinant bacterial immunogens, a variety of factors minimize this potential. Not only can adverse effects occur from BCG especially in the immunoincompetent individual, but also routine use in the United States could affect the epidemiologic significance of a reactive tuberculin test. Previous tuberculin exposure can blunt response to a mycobacterial recombinant. It is possible, however, that further attenuated BCG strains (or other mycobacteria) could minimize opportunistic complications and antigenic manipulations might diminish tuberculin cross-reactivity. Recombinant Commensal Bacteria. A variety of natively avirulent (or virtually so) bacteria could be used as substitutes for attenuated pathogens in producing recombinant immunogens. Data are being accumulated that the introduction of recombinant commensals into a normal flora setting can induce an immune response and more information has been derived regarding the genetics of some of these organism^.'^ One example of this approach is a recombinant Lactococcus lactis engineered to express fragment C of tetanus toxoid.'j6When given to mice intranasally, serum and secretory antibodies were produced. Protection against tetanus was demonstrable even without the need for invasion or even colonization of the nasal mucosa. Another system utilized recombinant baker 's yeast, Saccharomyces cerevisiue, in which epitopes of hepatitis B surface antigen were expressed on the yeast cell As final examples, Sfreptococcus gordonii, a component of human mouth flora, has been made to produce the E7 protein of human papilloma virus type 16. A single intravaginal inoculation of the organism given to mice intravaginally resulted in both local IgA and serum IgG anti-E7 antib0dy.5~The same group subsequently immunized primates similarly with S. gordonii expressing either E7 or the V3 domain of HIV-1 gp120 and elicited vaginal IgA, serum IgG, as well as a T-cell proliferative response.20 Vaccinia and Other Poxviruses. The vaccinia virus immunization program successfully eradicated smallpox using an immunogen that was potent as a single dose, convenient to use in the field, stable without refrigeration, and cheap. The virus has a large capacity for foreign genes and the ability efficiently to express multiple foreign proteins and induce both humoral and cellular immunity.17 The well-documented, albeit uncommon, complications of standard vaccinia use, however, has made

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the first step in this technology to be more mycobacterial attenuation to produce a safe but still immunogenic biologic product. Two strains in particular have been developed that may well be acceptable on an attenuation basis for clinical trials. One strain, NYVAC, was derived from the Copenhagen strain of BCG by Tartaglia et a189by serial deletions of virulence-associated and host-range genes (18 in all). Another, modified vaccinia virus Ankara (MVA), was developed from serial passages in primary chick embryo fibroblast^^^ and has been used clinically as a smallpox vaccine in Germany.5O MVA is host-restricted, unable to complete replication in most mammalian cells, but it still retains the ability in the nonpermissive cell to produce large quantities of recombinant material under control of late ~ r o m o t e r sRecombinant .~~ W A C strains have been shown to be protective in animals, for example against pseudorabies virus in mice and swine.'O Likewise, MVA recombinants expressing parainfluenza virus 3 antigen were protective in mice.% Recombinant avipoxviruses have also been studied as vaccine vectors. These viruses are similar to MVA in that foreign proteins can be expressed in human cells despite a nonreplicative, abortive infection. Examples include canarypox recombinants protecting rabbits against rabbit hemorrhagic disease eliciting cellular immune responses against tumor antigens, such as carcinoembryonic and being safe and immunogenic expressing rabies glycoprotein in humans.29A canarypox recombinant is now being studied as an HIV vaccine.'4a Other Viral Systems. A variety of other recombinant viruses have also been studied as immunogens, with adenovirus being the most studied. Although as compared with poxviruses the amount of DNA that can be contained is somewhat small, insertions into the early gene regions E3 and E4 have been investigated. Recombinant adenoviruses expressing hepatitis B surface antigen (HBsAg) were able to replicate and an adenovirus expressing and elicit anti-hepatitis B in chimpa~~zees'~ rabies glycoprotein could induce immunity to rabies in animals.'O0 As with all of these recombinant vectors, the effect of pre-existing immunity against the vector must be assessed and boosters may be best effective with purified antigen alone. Investigation of herpesvirus (especially disabled infectious single cycle)58recombinants is also underway. NEW ROUTES FOR IMMUNOGEN DELIVERY

Modification of Injection System. The jet-gun technique is a needleless system developed for immunization by the US Army and first used in 1954. It can facilitate the rapid immunization of large numbers of individuals. In today's world of universal precautions to prevent the transmission of blood-borne viruses, the lack of risk from accidental sharps exposure could be a clear advantage of the technique. Equal or better serologic responses occur from these jet-gun injections as compared with those with the standard syringe-needle m e t h o d o l ~ g yIt. ~has ~

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GRADON&LUTWICK

been reported, however, with the use of multidose vaccine vials in conjunction with the jet-gun, that transfer of virus-containing blood through reflux from the nozzle can occur and transmit infection.12Additionally, a recent report emphasized a statistically higher incidence of local signs and symptoms relating to the technique.% A single-use jet-gun cartridge may offer a better alternative, at least related to the risk of blood-borne virus transmission. A multicenter studyUreported that tolerance was acceptable and this technique was of equal or superior immunogenicity, with trials performed in France and West Africa involving a variety of immunogens. More immediate and delayed reactions did occur, however, with the procedure as compared with standard techniques and were felt to be inherent to the injection technique itself. Epicutaneous Immunization. Intact skin represents a major barrier against infection by virtue of an external layer, the stratum corneum, through which even very small molecular moieties have difficulties transversing. Therefore, large antigenic structures administered on this intact cutaneous barrier remain there unless carried by an invading microbe. The skin’s role in protection against infection, however, is not just a physical one. DC, a differentiated epidermal, bone marrowderived, Langerhans’ cell, plays a major role in the immune system. DCs are quite effective 8-cell and T-cell lymphocyte stimulators. As antigenpresenting cells, they can stimulate quiescent, naive, and memory B and T cells; function as immunologic sentinels; and readily elicit helper and killer T cells, antibodies, and cytokines.6 In reality, the science of vaccinology has only just begun to appreciate and utilize the DC in improving immunologic responses to vaccines. These cells also play a role in immunologic tolerance and manipulation of them could assist in allergy, organ transplantation, and autoimmune diseases. Techniques have been recently evaluated to better target the DC. Certainly, direct intradermal administration of antigen has been used and can be a method to induce immune reactivity even if no response occurred after deeper administration. As an example, a subgroup of nonresponders to hepatitis B vaccination by subcutaneous= or intramusculaf*routes can be made anti-hepatitis B reactive by intradermal inoculation, Pre-exposure rabies vaccination is, in fact, commercially available using intradermal vaccination. This route, however, does not inevitably result in immunologic recognition. As an example, truly tuberculinnegative individuals do not become sensitized by Mantoux testing even when the same area of skin is used repeatedly or if large amounts of antigen are ~ s e d . 2 ~ Transepidermal delivery of antigens for processing by DCs now appears possible using a novel carrier, the transfersome. This ultradeformable entity, a combination of phosphatidylcholine, sodium cholate, and the detergent sodium dodecyl sulfate, can be produced in conjunction with immunogens with a diameter of 100 to 200 nm. It appears to be able to cross intact mammalian skin with an efficiency of close to 100%. The transfersome system, besides being used as a drug carrier, can clearly deliver antigens into the body for immunologic processing.

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In a recent studyn the utility of transfersome delivery (applied on manually hair-trimmed intact skin) using gap junction protein as an antigen was compared with the subcutaneous route. Epicutaneous gap junction protein delivery in transfersomes using experimental animals produced an antibody response marginally higher than subcutaneous gap junction protein injections in transfersomes, mixed lipid micelles, or liposomes. Only transfersomes were effective transdermally and this route appeared to increase relative concentrations of specific IgA antibody Recombinant vectors as immunogens have also had epicutaneous application. Tang et aP8 have successfully produced specific antibody responses by administering a recombinant adenovirus containing the carcinoembryonic antigen or GM-CSF genes as approximately lo8 plaque-forming units in a volume of 10 to 50 pL onto preshaven, depilatory-treated mouse skin. This suggests that such a methodology could be developed for use by individuals with no specialized training, equipment, or sharps. It is presumed that the vector enters through microscopic trauma to the skin, perhaps produced during the skin preparation. Microparticle Delivery Systems. Although coating microparticles with antigens can assist in enhancing an immune response by better or more prolonged delivery of the antigen to processing cells, the technology of microencapsulation can now provide a new system for antigen release. As reviewed by Morris et a1f2biogradable polymer microspheres may be able to lower the number of required immunizations and enhance an immune response from either parenteral or oral routes. Antigens encapsulated in microparticles seem to produce a more prolonged depot as compared with alum adjuvanted antigens.J3The immune response, in fact, may be comparable with that using Freunds complete adjuvant.68 Polymer particles may be produced with antigen (or antigens) interspersed throughout the microparticle (monolithic)or as a core of antigen with a polymer coat (reservoir). Particle size can range from 1 to 300 p.Particle size appears to be a key factor in the induction of the quality and quantity of the immune re~ponse.~ One of the most investigated polymers studied for sustained release is the well-tolerated combination of lactic acid and glycolic acid. Indeed, polylactide-co-glycolide (PLG)polymers have already had many years of utilization as absorbable suture material. The release of antigen from the PLG particle is primarily related to matrix degradation and not from diffusion through matrix pores, although an initial surge of antigen release through surface pores occurs in monolithic microspheres. The rate of release from the PLG polymer matrix, indeed, can be programmed by modifymg microsphere diameter, changing antigen load, copolymer molecular weight, or the ratio of the A 50:50 copolymer microsphere has a components in the half-life of 1 week, whereas 100% lactide or glycolide have half-lives of 5 to 6 months.6oMorris et ala also point out that even more flexibility can be produced by the combination of reservoir monolithic microspheres with different characteristics. Furthermore, ultrasound, magne-

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tism, light, and other physical modalities can control antigen rate by influencing polymer breakdown. An example of this technology is shown by Singh et a18" using encapsulated diphtheria toxoid in PLG polymer microspheres. In this study, the immunogenicity and challenge protection of combinations of different-sized monolithic diphtheria toxoid-PLG microparticles given once was comparable with a three-dose series of alum adjuvanted diphtheria toxoid. Using alum adjuvanted diphtheria toxoid in the microspheres had the best immunogenicity. Biodegradable microparticles can also be prepared to contain antigen and cytokines, which may further potentiate long-term immune responses.73Plasmid DNA has also been encapsulated in PLG polymer and has been effective as an [email protected] Microparticle technology has successfully demonstrated some protection against experimental challenge of human volunteers against enterotoxigenic E. c ~ l i . ~ ~ Other polymers studied include iminocarbonate polymers with added tyrosine, which seems to provide additional adjuvant effect independent of a sustained-release and the so-called biovector. The biovector, a nanoparticle made of polymerized polysaccharides, with substituted phosphate residues and bound with palmitic acid, appears to have good adjuvant-antigen delivery properties." Enhancing Mucosal Antigen Delivery. The surface barrier of the mucosae of the respiratory and gastrointestinal systems protects the body against a variety of toxic and infectious pathogenic threats. It also minimizes the uptake of antigens needed for a successful mucosal immunization. Such adequate mucosal vaccination, whether achieved intranasally, orally, or otherwise, can translate into both strong panmucosal and systemic immunity. Scattered amidst a sea of epithelial cells linked together with gasket-like intercellular tight junctions are M cells. These M cells sample the antigenic milieu of the luminal contents, transporting both soluble and particulate antigen across their depth to underlying immune-processing macrophages. The use of antigen-loaded microparticles can facilitate antigen delivery, but it has now been established in vitro that Peyer's patch lymphocytes can convert the lining enterocytes into functional M cells.42Increasing the M-cell population can translate into facilitating a more brisk immune response. The trigger for such conversion is not yet clear,Q 49 but cytokines are suspected to play a role. Elucidation of the factors in conversion is likely to improve even more the efficacy of mucosal immunization. Microspheres coated with UZex europareus 1 lectin have also been found to increase M-cell targetingz8 THE PENULTIMATE IMMUNOGEN? EDIBLE RECOMBINANT PLANTS

The production of an inexpensive, relatively heat-stable, edible immunogen contained in a fruit or vegetable now is achievable, combining

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recombinant DNA technology with mucosal-acquired immunity. It is this type of vaccinology that may bring much more effective immunization utility and availability to the developing world. Genetic manipulation of plants has already been successful in a variety of arenas in agriculture.'O' Plants have been produced that are resistant to insect infestations, viruses, as well as herbicides. Additionally, tomato shelflife has been prolonged by genetic inhibition of ethylene production and the protein, fatty acid, and starch content of vital crops can be altered. In 1992, Mason and colleaguess published a seminal report regarding the production, in tobacco plants, of HBsAg. The tobacco-derived recombinant HBsAg was found to self-assemble into typical HBsAg particles, which were essentially identical to human serum HBsAg in respect to particle size, sedimentation density, and antibody binding. These recombinant plants were produced from seed obtained from plants transformed by a recombinant Agrobucfevium tumefaciensbacterial infection. Agrobucfevium-mediated transformation is one of the most widely used approaches to create transgenic plants. Other bacteria, however, as well as recombinant plant viruses can be used.'o' The yield of rHBsAg was [email protected] in the tobacco plant with as much as 0.01% of total plant protein found to be in the form of HBsAg. Furthermore, the immunogenicity of the tobacco-derived viral antigen was found to mimic closely that of the commercial Saccharomyesyeast-derived hepatitis B virus vaccine with preservation of both Bcell and T-cell epitopes." Practically the use of the tobacco plant (Nicotiunu t ~ b ~ c u mas) a recombinant factory for immunogens is quite limited by the potential harmful effects of other tobacco-associated substances. Other systems and antigens have, however, been studied. As examples, E. coli enterotoxin, Nonvalk virus capsid protein, and rabies virus glycoprotein have been produced in a number of edible plants including potato, spinach, 52* 57, It seems to be the case that recombinant tomato, and lettuce.35* antigens that are assembled into a particulate form or are expressed on the surface of recombinant plant viruses can produce effective immune responses even in the absence of a coadministered mucosal adjuvant?' E. coZi enterotoxin, or a modified toxoid, may function as a mucosal adjuvant for other recombinant antigens in this regard. Several of these systems have progressed to the point of protective experiments in animals. Using transgenic potatoes that expressed the S protein of porcine transmissible gastroenteritis virus, the pigs fed transgenic tubers had lower morbidity and mortality from infection than controls." Likewise, both immunogenic and protective effects were found in mice fed raw potatoes expressing the B subunit of E. coli heat labile enter~toxin.~~ Similar data have been found in mice using a cholera toxin model? Protective effects can also be demonstrated for systemic infection. As an example, mice immunized with a rabies recombinant alfalfa mozaic virus produced in spinach were found to have amelioration of clinical signs produced by subsequent challenge by intranasal infection with an attenuated rabies virus strain?' Initial data are now available in humans utilizing the transgenic potato producing E. coli

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enterotoxin subunit. Tacket et a1%found that human volunteers fed such potatoes (50 to 100 g of raw potato delivering a mean of 0.75 mg antigen/dose) developed specific mucosal and systemic antibody with good tolerance. It is important to ensure that such food-borne immunization does not result in oral tolerance. Oral tolerance refers to the minimal or absent immune response to food components elicited from the mucosal immune response. Indeed, animals fed antigens usually have only a minimum response to the same antigen when given systemically3Such tolerance, if induced by this technology, could cause more disease rather than prevention and clearly must not be an issue prior to introduction of this vaccination strategy in man. It is also possible that coadministered 86 mucosal adjuvants would overcome or avoid toleran~e.~, The ultimate end for this technology is the use of an edible, highly productive plant (both in fruit and recombinant antigen) grown in the developing world. The banana, which can be produced at a cost of 1 to 2 US cents per fruit, seems to produce 0.1% to 1% foreign protein culminating in 1- to 10-mg recombinant protein per fruit. This could represent 1 to 10 or more doses per fruit. Because the time to produce such transgenic bananas is long, it will be some time prior to useful data in this system.

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Address reprint requests to Larry I. Lutwick, MD Infectious Diseases (HIE) VA Medical Center 800 Poly Place Brooklyn, NY 11209