Vaccines: Protective Immunizations by Parenteral, Mucosal, and Gene-Gun Inoculations
Vaccines: Protective Immunizations by Parenteral, Mucosal, and Gene-Gun Inoculations
Vaccines: Protective Immunizations by Parenteral, Mucosal, and Gene-Gun Inoculations
USA
Vol. 90, pp. 11478-11482, December 1993
Immunology
AND HARRIET L.
*Department of Pathology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655; tDepartment of Virology and
Molecular Biology, St. Jude Children's Research Hospital, 332 North Lauderdale, P.O. Box 318, Memphis, TN 38101-0318; and tAgacetus Inc., 8520
University Green, Middleton, WI 53562
ABSTRACT
Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to
raise protective immunity against lethal influenza challenges of
the same subtype. In trials using two inoculations of from 50 to
300 pg of purified DNA in saline, 67-95% of test mice and
25-63% of test chickens have been protected against a lethal
influenza challenge. Parenteral routes of inoculation that
achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far
the most efficient DNA immunizations were achieved by using
a gene gun to deliver DNA-coated gold beads to the epidermis.
In mice, 95% protection was achieved by two immunizations
with beads loaded with as little as 0.4 pg of DNA. The breadth
of routes supporting successful DNA immunizations, coupled
with the very small amounts of DNA required for gene-gun
immunizations, highlight the potential of this remarkably
simple technique for the development of subunit vaccines.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. 1734 solely to indicate this fact.
11478
11479
11480
Table 1. Protection of mice against a lethal A/PR/8/34 (HlNl) influenza virus challenge by inoculation of pCMV/H1
DNA in saline
Route of
Dose,
Signs of
No. of survivors/
DNA
inoculation*
pg
influenza
no. tested
% survival
Probability
++
300
pCMV/H1
i.v., i.p., i.m.
21/22
95
<0.0001
++
i.m.
200
18/19
95
<0.0001
++
i.v.
100
10/12
83
<0.0001
+++
i.n.
100
13/17
76
<0.0001
++++
i.d.
50
9/12
75
<0.001
++++
s.c.
100
4/6
67
<0.02
+++++
i.p.
100
0/6
0
++ ++ +
Various
0-300
pCMV/control
3/24
13
See Materials and Methods for details on vaccination trials. Signs of influenza included weight loss, ruffled fur, and
lethargy. These were scored as follows: +, transient weight loss but maintenance of smooth fur and normal levels ofactivity;
+ +, transient weight loss, some ruffling of fur and lethargy; + + +, transient weight loss and more severe ruffling of fur
and lethargy; + + + +, more prolonged weight loss coupled with severe ruffling of fur and lethargy; + + + + +, weight loss
and severe signs of influenza leading to death. Data are pooled from four independent trials. No data for the reported
conditions have been omitted. Probability was calculated by using Fisher's exact two-tailed test comparing the frequency
of survival and mortality in vaccine versus control groups.
*i.v., Intravenous; i.p., intraperitoneal; i.m., intramuscular; i.n., intranasal; i.d., intradermal; s.c., subcutaneous.
Table 2. Protection of chickens against a lethal A/Chicken/Victoria/1/85 (H7N7) influenza virus challenge by
inoculation of H7-expressing DNAs in saline
Route of
No. of survivors/
Dose,
% survival
DNA
inoculation*
no. tested
pg
Probability
<0.0001
300
50
p188
i.v., i.p., s.c.t
28/56
24
<0.01
i.v.
100
8/33
0
100
i.p.
0/8
0
s.c.
100
0/2
2
1/55
300
pRCAS
i.v., i.p., s.c.t
<0.0001
i.v., i.p., i.m.
63
300
pCMV/H7
19/30
<0.02
30
200
i.m.
3/10
30
<0.02
i.t.
200
3/10
10
i.b.
200
1/10
10
200
i.o.
1/10
2
0-300
Various
pCMV/control
1/43
See Materials and Methods for details of vaccination trials. Within experimental groups, survivors showed varying signs
of influenza. Data for p188 and pRCAS DNA are pooled from five independent trials. Data for pCMV/H7 and
pCMV/control DNA are pooled from seven independent trials. No data have been omitted. Probability was calculated by
using Fisher's exact two-tailed test comparing the frequency of survival and mortality in vaccine versus control groups.
*i.t., Intratracheal; i.b., intrabursal; i.o., intraorbital; for others, see footnote to Table 1.
tData reported in ref. 4.
DISCUSSION
The results of our vaccine trials demonstrate that epidermal,
mucosal, intramuscular, and intravenous routes of administration can be used for DNA vaccines (Tables 1-3). Our
results also demonstrate that gene-gun delivery of DNA into
the epidermis is a very efficient method of inoculation,
achieving protection with 250-2500 times less DNA than
direct inoculations of purified DNA in saline (Tables 1 and 3).
Transfection Efficiency Versus Vaccination Efficiency. One
of the striking results of our DNA vaccine trials is that the
efficiency of transfection does not necessarily determine the
efficiency of vaccination. The high ability of rodent muscle to
take up and express DNA (5-7) did not correlate with an
unusual efficiency ofintramuscular vaccinations (Tables 1, 3,
and 4). In the mouse trials, intramuscular inoculations
worked well, but no better than intravenous inoculations, and
only somewhat better than the administration of DNA nose
drops (Tables 1 and 4). Similar results were obtained in the
chicken trials, where intravenous and intratracheal inoculations achieved levels of protection comparable with those
provided by intramuscular inoculations (Table 2).
The success of DNA immunizations by the intravenous and
mucosal routes may reflect efficient antigen presentation and
recognition compensating for inefficient transfection. Both
blood and mucosal surfaces have associated lymphoid tissues
that provide specialized and highly active immune surveillance. Thus, successful vaccination by these routes may
reflect a highly efficient response to very low numbers of
transfected cells.
High Efficiency of Gene-Gun Vaccinations. Highly efficient
immunizations were achieved by gene-gun delivery of DNA
to the epidermis of mice. This method of immunization
11481
A/PR/8/34 (HlNl)
DNA and route
Time of
bleed
No.
tested
Prevac
10 d PB
4 d PC
14-19d PC
Prevac
10 d PB
4d PC
14-19 d PC
Prevac
10 d PB
4dPC
14-19d PC
2 (12)
2 (12)
1 (6)
2 (10)
3 (19)
3 (19)
2 (13)
3 (18)
3 (17)
3 (17)
ELISA value x
10-2
HI
pCMV/Hl
in saline
i.v.
i.m.
i.n.
2(11)
3 (17)
<
<
<
<
<
<
<
8
128
256
<
<
<
<
3
4
4
4
<
<
32
406
<
<
<
<
<
<
<
<
1
160
2
2
202
1
1
2
20
113
<
<
6
127
pCMV/control
in saline
<
<
<
<
Prevac 3 (16)
<
<
<
<
10 d PB 3 (16)
<
<
<
<
4 d PC 2 (9)
<
256 <
320
14-19 d PC 1 (2)
<
<
<
<
Prevac 2 (10)
pCMV/Hl,
10*
1
10 <
10 d PB 3 (16)
genegun
<
64
20*
2
4d PC 3 (16)
645 <
14-19 d PC 3 (15) 160* <
<
<
<
<
Prevac 2 (12)
pCMV/control,
<
<
<
1
10 d PB 3 (16)
gene gun
<
<
<
2
4dPC 3(16)
NT
4
512 <
14-19 d PC 1 (3)
Data are the geometric means of the reciprocal of the final dilutions
of pooled sera that scored positive for a given condition. Prevac,
bleed before DNA vaccinations; 10 d PB, sera harvested 10 days after
the second DNA inoculation, immediately prior to challenge; 4 d PC,
sera harvested at 4 days postchallenge. No. tested, no. of groups for
which pooled sera were assayed (total no. of animals contributing
sera to the pools); <, activity not detected in the lowest dilution of
serum used in tests [1:10 for hemagglutinin inhibition (HI); 1:100 for
ELISA]; NT, not tested.
*Only one of the three pools of sera was tested for HI activity.
Various
required 250-2500 times less DNA than the saline inoculations (0.4-0.04 ,ug as opposed to 100-200 p,g of DNA) (Tables
1 and 3). We think the remarkable success of gene-gun
vaccinations reflects the combination ofefficient transfection
with efficient antigen presentation and recognition. The gene
gun represents a very effective method of transfecting a
tissue (24, 26). When the epidermis is transfected, DNAexpressed antigens are subject to immune surveillance by the
skin-associated lymphoid tissue. This lymphoid tissue is rich
in cells (such as epidermal Langerhans cells) that are capable
of presenting transfected antigens to the T-helper component
of the immune system (12-14, 27).
Induction of Memory by DNA Vaccinations. DNA immunizations rely on low numbers of transfected, antigenexpressing cells to raise immune responses. In our trials,
these low numbers of antigen-expressing cells did not induce
high-titer antibody responses (Table 4 and ref. 4). However,
they did prime both T-helper and B-cell memory. This
memory appeared to provide protection by supporting the
mounting of secondary responses in challenged animals (Table 4). Evidence for the priming of memory is provided by the
DNA inoculations raising antibodies belonging to the IgG
11482