NIH Comments on

FDA's Draft Guidance for Industry:

Considerations for Plasmid DNA Vaccines for

Infectious Disease Indications

Docket \# 2005D-0047

May 18, 2005

The National Institutes of Health (NIH) has the following comments on
the *Draft Guidance for Industry: Considerations for Plasmid DNA
Vaccines for Infectious Disease Indications*. NIH appreciates FDA's
interest in gathering input on the draft document. These comments
reflect perspectives, in particular, of the National Institute of
Allergy and Infectious Diseases (Division of AIDS and the Vaccine
Research Center) and the National Cancer Institute (Division of Cancer
Biology/Cancer Etiology Branch), that are based on a broad experience in
the development of DNA vaccines, including nearly two dozen DNA vaccine
candidates for prophylactic indications (e.g., for HIV/AIDS, Ebola,
SARS, and WNV), 13 of which have reached the clinic.

First, the guidance seems primarily directed to products going into
Phase 1 clinical development as would be appropriate given that most DNA
plasmid vaccines are in that phase of development currently. However,
with the exception of the discussion on potency, it is not clear whether
the guidance would apply to all phases of clinical development. It would
be helpful to clarify whether later phase clinical development studies
should be taking different approaches and whether there are plans to
develop specific guidance for later phase studies.

Second, the guidance does not address some of the key issues that
investigators, industry and funding agencies are currently considering
and on which guidance would be helpful. These include:

-   Are measurements of residual antibiotic in the product needed? There
    are difficulties in measuring residual antibiotic in the presence of
    high concentrations of plasmid DNA, and, although FDA has asked
    sponsors about this issue, it is not clear whether FDA expects such
    measurements to be taken for plasmid DNA vaccines. It would be
    helpful if the guidance indicated in Section II whether or not an
    effort should be made to measure the presence of residual antibiotic
    and, if so, how such measurements can be accomplished.

-   Modifications to the method and route of administration of the
    plasmid vaccine. In addition to guidance on modifications to the
    sequence of the DNA plasmid vaccine, it would be helpful to have
    guidance on modifying the method and route of administration of the
    vaccine. Specifically, what would be the preclinical biodistribution
    and toxicology requirements to proceed to phase I testing where
    there may be previous human experience or extensive preclinical
    safety experience using the same method or route to deliver
    different, but similar, plasmids.

-   Testing prime-boost regimens. It would be helpful to know whether it
    is necessary to test prime-boost regimens in repeated-dose
    toxicology studies (as opposed to testing the individual components
    in separate studies). We have not found pre-clinical toxicity of
    prime-boost regimens to be synergistic and toxicities identified for
    the individual components were the same as those seen in prime-boost
    toxicology studies. As such, we would suggest that the Guidance
    should not require prime-boost repeated-dose toxicology studies in
    most cases.

-   A section on clinical issues would be beneficial. Guidance on the
    following specific clinical issues, for example, would be helpful:

    -   How long after last vaccination must contraceptive use/safe sex
        practices be maintained to ensure safety? In addressing this
        issue, please consider the following:

        -   Reproductive toxicology has not been performed on most
            products in early clinical development.

        -   Clinical trials can last for a year or more, even Phase 1
            studies, and pregnant/lactating women are generally
            excluded. Maintaining contraceptive use would require most
            prospective female subjects to forgo having a child for at
            least two years (i.e., more than 9 months for pregnancy
            prior to the trial + however many months of lactation prior
            to the trial + the time on trial during which pregnancy must
            be avoided). For healthy young women, this is a long time
            and their decisions about family planning may change over
            time.

        -   From a safety standpoint, biodistribution of the DNA plasmid
            vaccine to the gonads is not actually seen for most vaccines
            delivered intramuscularly.

    -   Should subjects with allergies to antibiotics that may be
        present in trace residual amounts in the final product be
        excluded from the study? In addressing this issue, please
        consider the following:

        -   Since many of the plasmid vaccines contain the kanamycin
            resistance gene, kanamycin is used during fermentation.
            Although the purification methods are expected to remove
            residues, an assay for residual levels is technically
            infeasible because of interference (as noted above).

        -   Kanamycin is not generally used clinically, and our
            clinicians report that allergies to aminoglycosides are
            quite rare.

        -   Information about allergies to aminoglycosides cannot be
            reliably determined by patient history.

        -   Since clinical trial procedures generally require subjects
            to remain in the clinic for 15-30 minutes after vaccination
            in the rare event of anaphylaxis, it is not clear that
            screening subjects for allergies to residual antibiotics
            enhances subject safety.

    -   Is monitoring for auto-antibody production in the clinical phase
        still necessary? Guidance in Section IV (C) on autoimmunity
        suggests that FDA may no longer expect such monitoring during
        clinical studies. Please clarify whether this is in fact the
        case.

Third, flexibility would be helpful regarding Section II (B) on bulk
plasmid release testing (i.e., not the final formulated bulk immediately
prior to filling, which is generally considered as part of the final
product release).

-   Tests formerly recommended for final containers (i.e., in the 1996
    guidance) have now been moved to the bulk. The Guidance should allow
    flexibility about whether tests should be of the bulk or whether
    they should remain as tests of the final containers or final
    formulated bulks. Specifically, the tests for endotoxins or
    pyrogenic substances are generally expected to be performed on the
    final containers \[21 CFR 610.13(b)\].

-   The Guidance should be flexible about whether tests of potency are
    required on the bulk or the final formulated bulk/containers. Some
    tests of potency, such as expression, may be appropriate at the
    unformulated bulk stage, but if bioassays (i.e., *in* *vivo*
    immunogenicity) are expected as product development advances,
    testing potency on the unformulated (and possibly monovalent) bulk
    might not be appropriate.

Fourth, in the final sentence of the first paragraph of Section IV (C),
the Guidance does not distinguish between autoimmunity associated with
anti-dsDNA antibodies and other forms of autoimmunity. Although it
states that pre-clinical studies to assess "whether vaccination causes
autoimmune disease" are no longer expected, a paragraph later it refers
to concerns about autoimmunity in some cases (e.g., plasmids encoding
self-antigens). If there are lingering concerns about autoimmunity in
specific cases, it may make more sense to end the sentence with "whether
vaccination induces anti-dsDNA antibodies" rather than "autoimmunity."

Fifth, regarding Section IV (E), it would be clearer in the first
sentence to delete the phrase \"When appropriate and where possible\"
and begin the text with \"We encourage animal challenge. . . .\" Since
this testing is encouraged and not mandated, the inclusion of the
initial phrase unduly weakens the safety aspects without providing
examples or criteria of what is \"appropriate and possible.\"

Sixth, regarding Section IV (G) regarding the level of residual
biodistribution at "the site of injection,"

-   Guidance on residual biodistribution levels in terms of copies of
    plasmid/microgram of DNA extracted from the tissue rather than in
    copies of plasmid/number of cells. While extraction of DNA from
    tissue culture cells or blood cells may be determined based on
    number of cells, these biodistribution studies are being performed
    on tissue for which weight (grams) rather than cell numbers is
    measured. Generally, a certain amount (weight) of DNA is used per
    PCR reaction and, thus, reporting copies number per weight of
    extracted DNA is more logical and direct than relating the copy
    numbers to numbers of cells. While there is a direct relationship
    between weight of DNA extracted and number of cells, the number of
    cells is generally not measured from a tissue specimen, whereas
    weight of DNA extracted is, making it the logical unit for reporting
    the denominator in biodistribution studies.

-   For intramuscular injection, please clarify whether you are
    referring to the muscle or including the overlying subcutis and skin
    as part of the "site of injection."

-   The value indicated as persisting at 60 days in the site of
    injection (based on published literature) may be the experience when
    DNA plasmid vaccines were given at doses such as 100 micrograms in
    mice. However, when multiple milligrams of DNA are given (as they
    currently are), residual levels at the muscle are often present at
    the level of 100's or even 1000's of copies/microgram of DNA at day
    60 in a minority of animals. Moreover, if the subcutis/skin is
    considered, sometimes a majority of animals still have PCR signals
    at day 60, and these are generally on the order of 100's or 1000's
    of copies/mcg of DNA but can be up to 10^5^ or 10^6^ in a rare
    animal. A manuscript of some of these data (from the VRC vaccine
    candidates) is currently being prepared for submission for
    publication. However, the experience described herein also includes
    what has been seen for DAIDS-sponsored DNA plasmid vaccines as well,
    many of which have already been permitted by FDA/OVRR to proceed
    into clinical studies on the basis of such data. As such,

    -   Please reconsider the dataset and determine how applicable it is
        to the current clinical situation when multiple milligrams of
        DNA are being delivered rather than micrograms of DNA.

    -   Please clarify whether the value cited in the Guidance
        specifically refers, for intramuscular injection, to levels in
        the muscle, which are lower and present in a minority of animals
        at day 60, or including the subcutis/skin, which are higher and
        more consistent (i.e., sometimes present in a majority of
        animals).

    -   Please cite the published studies referenced in the Guidance.

We would like to thank the FDA for providing updated guidance on this
platform technology and seeking input on it. We appreciate the
opportunity to share our perspectives.