Anthrax Vaccine by Patch articles

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http://www.jci.org/cgi/content/full/114/7/868

J. Clin. Invest. 114:868-869 (2004).

Copyright ©2004 by the American Society for Clinical Investigation

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News

Taking the sting out of the anthrax vaccine

Laurie Goodman

Staff Sergeant , of the Boone, Iowa, National Guard Reserve,

is ready to do his duty when it comes time for members of his unit to get

the next of six shots given over an 18-month period for anthrax

vaccination. Although he is not entirely happy about it, he told the JCI,

" I really don’t have another choice other than get out, and I’m not

prepared to do that. " He said he simply hopes he doesn’t have another

adverse reaction. Soon after his third shot, suffered leg cramps

caused by blood clots. " We can’t really pinpoint the real reason I came

down with this, " he noted, " other than it’s just a coincidence that I

happened to get the shots and then came down with this. But I think the

military, or whoever is building this vaccination, should follow up a

little more with the people who had issues with it — to see if it did in

fact have a reason for it, or was a problem at all, or why it happened. "

While remains equivocal about the relationship between his health

issues and the anthrax vaccine that is currently mandatory for military

personnel considered to be at risk for biological weapons offensives,

others, such as retired US Air Force Reserve pilot Lieutenant Colonel Jay

Lacklen, who was stationed at Dover, Delaware, are adamant that the vaccine

is to blame for myriad problems, primarily autoimmune responses that

resulted in symptoms of extreme vertigo, intense muscle and joint pain, or

mental impairments and ailments. Lacklen said that the occurrence of one of

" those three [types of symptoms] almost immediately after the series of

shots started at Dover caused 40% of our reserve pilots to leave the unit

rather than take the shot. "

A great deal of controversy has surrounded the use of the anthrax vaccine,

including speculation as to whether it is the cause of Gulf War syndrome.

There have been numerous accusations and investigations, and while the

vaccine is still approved for mandatory military use, the uproar regarding

its overall safety continues (see http://www.milvacs.org and

http://www.anthrax.osd.mil for more information). The vaccine at the center

of this storm uses a cell-free filtrate — a mix of dead bacteria as opposed

to live bacteria — to stimulate the appropriate immune response. Only

BioPort Corp. manufactures it; that fact, along with its history of having

repeatedly failed FDA inspections, has further stirred the furor over its

mandatory use among military personnel.

Now come the next generation of anthrax vaccines, which are based on the

use of recombinant protective antigen (rPA), a main component of anthrax

exotoxin, to stimulate the protective immune response. Such a vaccine is

already in phase II clinical trials.

Interest, however, in developing a needle-free anthrax vaccine is high. Two

recently studied vaccines, one applied via a skin patch (1) and one that is

inhaled (presented at the 228th National Meeting of the American Chemical

Society in August; see

http://oasys2.confex.com/acs/228nm/techprogram/P784204.HTM for abstract),

show promise in their ability to protect against anthrax in preliminary

animal studies.

" The [development of] non-needle vaccines is a whole fascinating field, "

Belshe told the JCI. Belshe, who is the director of the Center for

Vaccine Development at Saint Louis University and helped develop FluMist, a

flu vaccine nasal spray, highlighted the importance of making these

vaccines pain-free, since a " substantial proportion of the adult population

won’t get their influenza shot because it’s a needle and they’re afraid of

it. " He noted that there were other issues that make the development of

needle-free vaccines even more worthwhile. For example, " for developing

nations, where they may be reusing needles or boiling needles, there is

always the question, are you transmitting hepatitis or AIDS or something

through a parietal injection? "

Matyas, of the Walter Army Institute of Research, who coauthored

the paper on the development of a skin-patch version of the anthrax vaccine

(1), added that a patch system " is much easier to administer. You don’t

need much training to give it, and we should be able to deploy it into the

field. "

Belshe and Matyas both pointed out, however, that the major advantage of

needle-free vaccines is that they directly target places where most

infections initiate. Noel Harvey, director of Advanced Drug Delivery at BD

Technologies and head of the group developing the inhaled anthrax vaccine,

agreed, explaining that " the development of vaccines [that] can be rubbed

onto the skin or placed into the nasal mucosa or the pulmonary mucosa is

really undertaken, not so much to avoid using a syringe, but to actually

get a mucosal response, in the case of intranasal or pulmonary delivery, or

to access the Langerhans cells and dendritic cell-type precursors in the

epidermis, in the case of delivery through the skin. "

Both the skin-patch version and the inhaled version of the anthrax vaccine

do use rPA to stimulate a protective response but are in preliminary

animal-testing stages.

Matyas and his colleagues at Walter , in a joint venture with IOMAI

Corp., have tested the anthrax patch on mice, immunizing them at 0-, 2-,

and 4-week intervals with a gauze pad soaked with rPA and differing amounts

of heat-labile enterotoxin (HLT) from E. coli as an adjuvant. At every

level of HLT, the mice showed 100% protection against anthrax (Sterne

strain) challenge. Matyas told the JCI that although this work was done

using HLT, " part of the research effort that we are doing here is to look

at other adjuvants, " and it is of note that rPA alone, without any

adjuvant, also afforded 100% protection (1).

In the inhaled-vaccine studies at BD Technologies, which are being

conducted in collaboration with the US Army Medical Research Institute of

Infectious Disease, the nasal cavity is targeted. The vaccine formulation

utilizes rPA with a mucosal adherent called chitosan. " We are very early in

this research and we are very encouraged with the findings of protection in

rabbits with a relatively simple powder formulation of recombinant

protective antigen, " Harvey said. " In those formulations with no other

additives that could be termed adjuvants [beside the mucoadhesive and CpG],

we did achieve protection of 100%. "

The finding that rPA alone might stimulate a strong protective response may

be good news for many in the military, since a great deal of the

controversy over the safety of the current BioPort anthrax vaccine centers

on the effect of the adjuvant. While the BioPort vaccine uses aluminum

hydroxide — which is standard in many US vaccines — as an adjuvant, it has

also been found to contain trace amounts of squalene, an adjuvant that is

not approved for vaccine use in the US but is used in some European

vaccines. Squalene is known to cause autoimmune reactions when injected

into animals. Although many have discounted the trace amounts as too small

to cause the types of reaction some have experienced, this finding has

created even more concern over the use of the current vaccine.

Progress of these non-needle vaccines from preliminary stages to approval

for human use, however, is many years off. Standard vaccine approval

requires extensive clinical testing after animal testing is complete.

Belshe explained that an appropriate dosage for the antigen in the vaccine

is determined through a series of tests in small animals. " And then you go

through a process of evaluating the vaccines in humans. Typically, young

healthy adult volunteers are given the first dose of vaccine, and if it’s

ultimately going to be a childhood vaccine, then you move gradually into

younger and younger populations. Or if it’s targeted for older folks, you

gradually work into an older population. You do this stepwise in small

numbers of persons so that you minimize risk and yet achieve reasonable

milestones of understanding of what’s going on. "

A vaccine for anthrax, or any other deadly infectious agent, obviously

cannot ever be tested in a challenge study in humans. Harvey stated that,

for the anthrax vaccine, " the general next steps are to do dose-titration

steps, to see if there is an optimal dose range to capitalize on, then move

into larger studies with protective correlates of man, like the rabbit

model we have used, then into higher primates to assure ourselves that we

are going to obtain protection and that the vaccine is safe and doesn’t

stimulate any undue responses or have any side reactions associated with it. "

There would, however, be no real certainty of the vaccine being protective

in humans. Matyas did note, though, that there are ways to obtain a sense

of the protective capability of a vaccine. " You can assay it for toxin

neutralization titers. At least in rabbit models, neutralization titers

correlate with protection. In humans one would have to make that

assumption. But of course that is not proven. "

The vaccine would never be used in the general population, as are those for

measles and smallpox, but would be used only in at-risk populations.

Currently, that means people in the military, such as and

Jay Lacklen. While might be resigned to receiving such a vaccine and

accepting its risks, he said that he " would want to know what some of the

effects of it could be. And I would maybe want to know if someone has a

family history of something that could affect them by taking it. " Lacklen

remains suspicious, given what he has seen at Dover Air Force Base, and he

finds the military’s answers to his questions about the presence of

squalene in the current vaccine unconvincing. He believes that military

personnel are being used as test subjects. Lacklen told the JCI, " I don’t

think anthrax is that potent a weapon on the battlefield. I think the

entire anthrax hype is to run the vaccine. " He added that he would not be

convinced that a new vaccine was safe unless its chain of custody had been

closely monitored and it was then tested for the presence of squalene.

Protection from infectious toxins for these men and women is important, but

from their standpoint, assurance of the safety of the vaccine that should

protect them seems only fair.

Belshe believes that a lot of fascinating work is now going on in

the development of non-needle vaccines.

Matyas envisions that the skin-patch vaccine, packaged like a

Band-Aid, could be easily deployed where needed.

References

Matyas, G.R. et al. 2004. Needle-free skin patch vaccination method for

anthrax. Infect. Immun. 72:1181-1183.[Abstract/Free Full Text]

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http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=321625

Go to website for figures and tables

2004, American Society for Microbiology

Infect Immun. 2004 February; 72(2): 1181–1183.

doi: 10.1128/IAI.72.2.1181-1183.2004.

Needle-Free Skin Patch Vaccination Method for Anthrax

R. Matyas,1* Arthur M. Friedlander,2 M. Glenn,3

Little,2 Jianmei Yu,3 and Carl R. Alving1

Department of Membrane Biochemistry, Walter Army Institute of

Research, Silver Spring, land 209101 U.S. Army Medical Research

Institute of Infectious Diseases, Fort Detrick, Frederick, land 217022

Iomai Corporation, Gaithersburg, land 208783

*Corresponding author. Mailing address: Department of Membrane

Biochemistry, Walter Army Institute of Research, 503 Grant

Ave., Silver Spring, MD 20910-7500. Phone: (301) 319-9477. Fax: (301)

319-9035. E-mail: gary.matyas@....

Three immunizations of mice with recombinant protective antigen (rPA) by

transcutaneous immunization (TCI) induced long-term neutralizing antibody

titers that were superior to those obtained with aluminum-adsorbed rPA. In

addition, rPA alone exhibited adjuvant activity for TCI. Forty-six weeks

after completion of TCI, 100% protection was observed against lethal

anthrax challenge.

Transcutaneous immunization (TCI) is a procedure that relies on application

of antigen and associated adjuvant onto the outer layer of the skin and

subsequent delivery to underlying Langerhans cells that serve as

antigen-presenting cells (4). A variety of adjuvants have shown

effectiveness in stimulating immunity by TCI, but the most widely used and

most effective adjuvants have been members of the ADP-ribosylating

bacterial exotoxins, such as cholera toxin or heat-labile enterotoxin (HLT)

from Escherichia coli (13).

Two days prior to immunization, hair was shaved on the backs of female A/J

mice (12 weeks old) purchased from the Laboratory (Bar Harbor,

Maine). Recombinant protective antigen (rPA; 20 µg) (Monoclonal

Antibody/Recombinant Protein Production Facility-Science Applications

International Corporation, National Cancer Institute-Frederick Cancer

Research and Development Center) was mixed with the indicated doses of HLT

in phosphate-buffered saline (purchased from Berna Biotech, Bern,

Switzerland) and applied to gauze patches placed on the backs of mice

overnight. The backs were shaved 1 to 2 days prior to application of the

patch. The site was hydrated with saline-soaked gauze and mildly abraded by

being brushed 10 times with emery paper (GE Medical Systems, Milwaukee,

Wis.) prior to patch application. Control groups consisted of HLT alone,

rPA alone, or intramuscular (i.m.) immunization with 10 µg of rPA mixed

with aluminum hydroxide (0.1 mg of Al3+ Rehydrogel HPA; Reheis Inc.,

Berkeley Heights, N.J.). Animals were bled, and sera were assayed for rPA

antibodies by enzyme-linked immunosorbent assay (ELISA), as described

previously (7, 9). Antiserum neutralization of Bacillus anthracis lethal

toxin cytotoxicity was determined by measuring the viability of 6 × 104

J774A.1 cells in the presence of lethal toxin (100 ng of PA/ml plus 50 ng

of lethal factor/ml) (10). At week 50, the mice were challenged

subcutaneously with 1,000 50% lethal doses (LD50) of B. anthracis Sterne

strain spores (1).

As shown in Fig. 1A, over a period of 47 weeks 100% of mice immunized with

rPA by TCI with HLT as an adjuvant responded with strong antibody titers

after applications of vaccine at 0, 2, and 4 weeks. All of the mice

responded briskly even after a single immunization. During the 43 weeks

after the last immunization the titers initially rose further and then

declined gradually (less than a log) to the end of the observation period.

A similar pattern, except for a more rapid rise in titer at 4 weeks, was

obtained after control i.m. injection with rPA adsorbed to aluminum

hydroxide (Fig. 1B).

At the end of 47 weeks, ELISA antibody titers of mice that received

different amounts of HLT as an adjuvant were measured (Fig. 2A). Maximal

adjuvant activity of the HLT was reached even with 0.4 µg of HLT, and the

antibody titer was not significantly increased with higher amounts of HLT.

Figure 2A also illustrates the interesting finding that rPA by itself, not

previously known as an adjuvant for inducing antibodies, had significant

activity without additional adjuvant for induction of antibodies to PA by

TCI. The strength of immunization with rPA alone was less than that

observed after TCI with rPA combined with HLT as an additional adjuvant,

and as shown in Fig. 2B, neutralizing antibody titers were much lower when

HLT was not present. Neutralizing antibody titers of aluminum-adsorbed rPA

were also much lower than those observed after TCI with rPA and HLT.

As shown in Table 1, at every level of HLT employed, TCI resulted in 100%

protection 46 weeks after the last immunization following lethal challenge

at week 50 with B. anthracis spores. The protection by TCI was identical to

the protection observed after i.m. injection with aluminum-adsorbed rPA,

which served as a positive control. The negative-control group that

received no immunization confirmed that the challenge employed was 100%

lethal. It is noteworthy that immunization with rPA alone, where rPA was

used as an antigen without additional adjuvant, also resulted in 100%

protection. From these data it is evident that, under the conditions

employed, TCI resulted in protective immunity that was at least equivalent

to that obtained after i.m. immunization with aluminum-adsorbed antigen.

Furthermore, if neutralizing antibody titers were important for protection,

as previously suggested (12), TCI would be expected to be a stronger

immunization strategy. These data are consistent with the previous

observation that anti-PA immunoglobulin G (IgG) is a significant in vitro

correlate of survival after lethal challenge with inhalational anthrax (10).

The present anthrax vaccines that are licensed in the United States and

United Kingdom have been subjected to criticisms for numerous real or

perceived suboptimal features and for frequency of adverse events (8). The

vaccines are incompletely characterized and are also difficult to

characterize; they are locally reactogenic like other aluminum-containing

vaccines, and the dose schedule is long (2, 8). Aluminum adjuvants have the

limitations of being associated with occasional severe local reactions such

as erythema, IgE induction, contact hypersensitivity, and granulomatous

inflammation, and they are not biodegradable and remain at the site of

injection for up to a year (6). Subcutaneous nodules that can last for

weeks are often found after subcutaneous injection of aluminum-containing

vaccines, including the U.S. licensed anthrax vaccine (6, 11). Although

aluminum adjuvants are usually viewed as relatively safe, large-scale

vaccination might gain better acceptance if a less reactogenic potent

adjuvant were used along with an improved immunization strategy.

The TCI vaccine strategy proposed in this study utilizes a recombinant

protein (rPA), lacks aluminum adjuvant, is administered without injection,

uses a potent adjuvant (HLT) that has a good safety record when

administered with TCI in humans (3, 5), induces specific antibody titers

that are at least equivalent to those observed after i.m. injection of

aluminum-adsorbed rPA, and causes long-lived (at least 47 weeks) protective

immunity against lethal (1,000-LD50) anthrax challenge.

Acknowledgments

We acknowledge the excellent technical assistance of Elaine on for

her work with the laboratory animals.

Research was conducted in compliance with the Animal Welfare Act and other

federal statutes and regulations relating to animals and experiments

involving animals and adheres to principles stated in the Guide for the

Care and Use of Laboratory Animals, National Research Council publication,

1996 edition.

This work was performed under a ative Research and Development

Agreement between Walter Army Institute of Research, Silver Spring,

Md., and Iomai Corporation, Gaithersburg, Md. Funding for the study was

provided by the Biological Defense Research Program, U.S. Army Medical

Research and Materiel Command, Fort Detrick, Md., and Iomai Corporation.

The information contained herein reflects the views of the authors and

should not be construed to represent those of the Department of the Army or

the Department of Defense.

Footnotes

Editor: A. D. O'Brien

1.Fellows, P. F., M. K. Linscott, B. E. Ivins, M. L. M. Pitt, C. A. Rossi,

P. H. Gibbs, and A. M. Friedlander. 2001. Efficacy of a human anthrax

vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by

Bacillus anthracis isolates of diverse geographical origin. Vaccine

19:3241-3247. [PubMed][Full Text]

2.Friedlander, A. M., S. L. Welkos, and B. E. Ivins. 2002. Anthrax

vaccines. Curr. Top. Microbiol. Immunol. 271:33-60. [PubMed]

3.Glenn, G. M., D. N. , X. Li, S. el, A. Montemarano, and C. R.

Alving. 2000. Transcutaneous immunization: a human vaccine delivery

strategy using a patch. Nat. Med. 6:1403-1406. [PubMed][Full Text]

4.Glenn, G. M., R. T. Kenney, L. R. Ellingsworth, S. A. Frech, S. A.

Hammond, and J. P. Zoeteweij. 2003. Transcutaneous immunization and

immunostimulant strategies: capitalizing on the immunocompetence of the

skin. Expert Rev. Vaccines 2:253-267. [PubMed][Full Text]

5.Güereña-Burgueño, F., E. R. Hall, D. N. , F. J. Cassels, D. A.

, M. K. Wolf, Z. J. , G. V. Nesterova, C. R. Alving, and G. M.

Glenn. 2002. Safety and immunogenicity of a prototype enterotoxigenic

Escherichia coli vaccine administered transcutaneously. Infect. Immun.

70:1874-1880. [ Free Full text in PMC]

6.Gupta, R. K. 1998. Aluminum compounds as vaccine adjuvants. Adv. Drug

Delivery Rev. 32:155-172.

7.Iacono-Connors, L. C., S. L. Welkos, B. E. Ivins, and J. M. Dalrymple.

1991. Protection against anthrax with recombinant virus-expressed

protective antigen in experimental animals. Infect. Immun. 59:1961-1965. [

Free Full text in PMC]

8.lenbeck, L. M., L. L. Zwanziger, J. S. Durch, and B. L. Strom (ed.).

2002. The anthrax vaccine. Is it safe? Does it work? National Academy

Press, Washington, D.C.

9.Matyas, G. R., and C. R. Alving. 1996. Protective prophylactic immunity

against intranasal ricin challenge induced by liposomal ricin A subunit.

Vaccine Res. 5:163-172.

10.Pitt, M. L. M., S. F. Little, B. E. Ivins, P. Fellows, J. Barth, J.

Hewetson, P. Gibbs, M. Dertzbaugh, and A. M. Friedlander. 2001. In vitro

correlate of immunity in a rabbit model of inhalational anthrax. Vaccine

19:4768-4773. [PubMed][Full Text]

11.Pittman, P. R. 2002. Aluminum-containing vaccine associated adverse

events: role of route of administration and gender. Vaccine 20:S48-S50.

12.Reuveny, W., M. D. White, Y. Y. Adar, Y. Kafri, Z. Altboum, Y. Gozes, D.

Kobiler, A. Shafferman, and B. Velan. 2001. Search for correlates of

protective immunity conferred by anthrax vaccine. Infect. Immun.

69:2888-2893. [PubMed][Free Full Text]

13.Scharton-Kersten, T., J. Yu, R. Vassell, D. O'Hagan, C. R. Alving, and

G. M. Glenn. 2000. Transcutaneous immunization on the skin with bacterial

ADP-ribosylating exotoxins, subunits and unrelated adjuvants. Infect.

Immun. 68:5306-5313. [PubMed][Free Full Text]

Figures and Tables

FIG 1. Time course of antibody titers to rPA in mice immunized (arrows)

with rPA by TCI (A) or i.

FIG 2. Serum IgG ELISA titers to rPA (A) and lethal toxin-neutralizing

antibody titers ( at week 47.

TABLE 1. Survival of mice challenged by subcutaneous injection with 1,000

LD50 of B. anthracis Sterne strain spores

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http://www.health.uab.edu/show.asp?durki=47341

UAB Microbiologists Investigate Novel Anthrax Vaccine

UAB microbiologists have been subcontracted by Vaxin, a Birmingham vaccine

development company, to develop an alternative anthrax vaccine that can be

administered topically through a skin patch. Funded by the National

Institutes of Health’s Small Business Innovation Research Program, the

project is led by principal investigators De-chu Tang, PhD, Kearney,

PhD, and Turnbough, PhD.

Dr. Tang explains, “Bacillus anthracis has three principal virulence

factors: protective antigen (PA), edema factor (EF), and lethal factor

(LF). Our hypothesis is that PA expression in the skin’s outer layer can

induce a systemic immune response against PA, thereby preventing EF and LF

from gaining access to target cells.”

Anthrax — an issue of concern even years ago — is the focus of research

being conducted by many at UAB.

For this project, a new generation of adenoviral vectors will be developed

as novel vaccine carriers. Dr. Kearney explains, “We will incorporate the

critical DNA segments of B anthracis into an adenovirus-based expression

vector, which we can grow in a cell line and harvest for application to the

skin.”

Dr. Turnbough calls the concept a “Band-Aid vaccine.” He says, “If

effective, it could be administered by wearing the patch for an hour,

making it much less expensive and easier to administer than the current

vaccine. Also, unlike the filtrate vaccine currently used, this recombinant

vaccine does not contain PA or other bacterial contaminants, which can

produce adverse effects.”

Until recently, the nation’s stockpile of anthrax vaccine was reserved for

the military and researchers in high-risk laboratories. No additional doses

have been manufactured since 1998, when the production line was suspended

following Food and Drug Administration citations.

Dr. Kearney comments, “In the event of a widespread anthrax outbreak, an

alternative vaccine would be invaluable.”

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UAB Synopsis, Vol. 21, No. 1, January 21, 2002

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http://www.stripes.com/01/oct01/ed101501e.html87

" Vaxin Inc., in Birmingham, Ala., announced Thursday it received a Small

Business Innovation Research Grant from the National Institutes of Health

to explore a skin patch to deliver the vaccine.

“The conventional anthrax vaccine requires at least six doses,” said Dr.

Kent Van Kampen, Vaxin president. “A vaccine patch could be applied by

anyone, not just medical professionals, and should not require six doses to

be effective.”

Vaxin said the skin-patch technology expands the capabilities of

researchers to create an entirely new and suitable anthrax vaccine, free of

adverse effects. In emergencies, this needle-free vaccination would not

require refrigeration or medical personnel for delivery.

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Sheri Nakken, R.N., MA, Classical Homeopath

http://www.nccn.net/~wwithin/vaccine.htm