WO2004043490A1 - Schema posologique pour vaccin antivariolique - Google Patents

Schema posologique pour vaccin antivariolique Download PDF

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WO2004043490A1
WO2004043490A1 PCT/US2003/035499 US0335499W WO2004043490A1 WO 2004043490 A1 WO2004043490 A1 WO 2004043490A1 US 0335499 W US0335499 W US 0335499W WO 2004043490 A1 WO2004043490 A1 WO 2004043490A1
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macaques
dryvax
vaccinia
virus
vaccine
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PCT/US2003/035499
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Genoveffa Franchini
James Tartaglia
Yvette Edghill-Smith
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Government Of The United States Of America As Represented By The Sercretary Of The Department Of Health And Human Services National Institutes Of Health
Aventis Pasteur, Limited.
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Priority to AU2003294248A priority Critical patent/AU2003294248A1/en
Publication of WO2004043490A1 publication Critical patent/WO2004043490A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/275—Poxviridae, e.g. avipoxvirus
    • A61K39/285—Vaccinia virus or variola virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens

Definitions

  • This invention relates to an improved method of immunizing against smallpox, hi particular, the method is useful in individuals with compromised immune systems, e.g., HIN- infected individuals and pregnant women.
  • smallpox vaccination was suspended because of its undesirable effects, particularly in some individuals, such as eczema patients.
  • eczema patients Much of the world' population has not been immunized against smallpox.
  • the risk of smallpox infection is believed to be increasing due to biological warfare stores of smallpox.
  • the side effects associated with current smallpox vaccines, e.g., Dryvax® still present a major obstacle in contemplating widespread vaccination to protect from smallpox.
  • MNA Modified Vaccinia virus Ankara
  • Vaccine regimens were tested in which MNA was administered as a priming vaccine, which was followed by administration of a standard smallpox vaccine, Elstree.
  • a serial vaccination (preliminary inoculation) using MNA was performed in primary vaccines with a variety of associated vaccine risks.
  • the initial vaccination with MVA was easily tolerated, allowing a second, mild cutaneous skin vaccination, which was performed without complication using a conventional vaccine (Stickl & Hochstein-Mintzel, Munc. med. Wschr.
  • MVA could be used to vaccinate irnmunosuppressed populations against smallpox (Mayr et al, Monbl Balcteriol B167:375-390, 1978; Mayr, A., Berl. Munch. Tierstoffitzl Klischr 112:332-328, 1999; and Mayr & Daniier Dev Biol Stand 41:225-234, 1978), as the virulence of MNA was not activated in immunosupressed host animals.
  • the current invention provides a method of inducing an immune response to smallpox.
  • the method comprises administering a highly attenuated vaccinia virus, such as NYVAC or MNA, in conjunction with a traditional smallpox vaccine virus, e.g., Dryvax, or the Elstree strain.
  • a highly attenuated vaccinia virus such as NYVAC or MNA
  • a traditional smallpox vaccine virus e.g., Dryvax, or the Elstree strain.
  • the method may be used for vaccinating immunocompromised individuals, e.g., individuals infected with HIN, individuals treated with immunosuppressive agents for cancer or organ transplant, individuals with congenital immunodeficiencies.
  • the vaccine regimen may also be administered to pregnant women.
  • Figure 1 A illustrates skin lesions induced by Dryvax in vaccinia-na ⁇ ve immune- competent macaques (Group 1).
  • Figure IB is a chart showing the mean lesion size obtained by imaging and time to resolution in macaques as illustrated in Figure 1 A (Group 1) (closed circles).
  • Figure 1C illustrates skin lesions in immune-competent macaques from Group 1 immunized again with Dryvax, approximately two months from the first Dryvax inoculation.
  • Figure ID illustrates skin lesions in macaques immunized one month before Dryvax challenge from Group la with one single intramuscular dose of NYVAC (1 x 10 8 pfu).
  • Figure IE is a chart showing the comparison of mean lesion size in macaques from
  • Group 1 (closed circles) and Group la (open triangles) over time.
  • Figure 2A is a chart showing the virus load of vaccinia-na ⁇ ve macaques infected with SHIN 89.6 PD (Group 2) (closed square: 3199; closed triangle: 3200; closed inverted triangle: 3222).
  • Figure 2B is a chart showing the CD4+ T-cell count of vaccinia-na ⁇ ve macaques infected with SHIN 89.6 PD (Group 2) (closed square: 3199; closed triangle: 3200; closed inverted triangle: 3222).
  • Figure 2C is a chart showing the virus load of vaccinia-na ⁇ ve macaques infected with SINmac251 (561) (Group 2) (closed square: 3149; closed triangle: 3176; closed inverted triangle: 3228).
  • Figure 2D is a chart showing the CD4+ T-cell count of vaccinia-na ⁇ ve macaques infected with SrVmac251 (561) (Group 2) (closed square: 3149; closed triangle: 3176; closed inverted triangle: 3228).
  • Figure 2E illustrates skin lesions in SHIN 89.6 PD-infected macaques (3222, 3199), and SINmac251 -infected macaques (AN3B, 3149, 3228) (Group 2).
  • Figure 2F is a chart showing the mean lesion size of macaques in Group 2 (open squares).
  • Figure 2G is a chart showing the comparison of mean lesion size over time in macaques from Group 1 (closed circles) and Group 2 (open squares).
  • Figure 3 A is a chart showing virus loads over time in macaques immunized with ⁇ YNAC before SINmac251 infection (closed circle: 11M; open square: 13M; closed triangle: 15M; open diamond: 20M).
  • Figure 3B is a chart showing CD4+ T-cell counts over time in macaques immunized with ⁇ YNAC before SINmac251 infection (closed circle: 11M; open square: 13M; closed triangle: 15M; open diamond: 20M).
  • Figure 3C is a chart showing virus loads over time in macaques immunized with ⁇ YNAC before SIVmac251 infection (open circle: R 7294; closed circle: RM7697).
  • Figure 3D is a chart showing CD4+ T-cell counts over time in macaques immunized with ⁇ YNAC before SINmac251 infection (open circle: RM7294; closed circle: RM7697).
  • Figure 3E is a chart showing virus loads over time in macaques immunized with MVA before SINsmE660 infection (closed square: H421; closed triangle: H456; closed inverted triangle: 18673).
  • Figure 3F is a chart showing CD4+ T-cell counts over time in macaques immunized with MNA before SINsmE660 infection (closed square: H421; closed triangle: H456; closed inverted triangle: 18673).
  • Figure 3G illustrates Dryvax-induced skin lesions in macaques previously immunized with ⁇ YNAC (Group 3).
  • Figure 3H is a chart showing the comparison of mean size of Dryvax-induced skin lesion between macaques previously immunized with ⁇ YNAC (Group 3) (closed triangle) and the vaccinia-na ⁇ ve immune-compromised macaques from group 2 (open square).
  • Figure 31 illustrates Dryvax-induced skin lesions in macaques previously immunized with MNA (Group 4).
  • Figure 3J is a chart showing the comparison of mean size of Dryvax-induced skin lesion between macaques previously immunized with MNA (Group 4) (closed inverted triangle) and the vaccinia-na ⁇ ve immune-compromised macaques from group 2 (open square).
  • Figure 4A is a chart showing Virus plasma levels in macaques immunized with ⁇ YVAC during an established SINmac251 infection (closed circle: 480; open circle: 644; closed inverted triangle: 680; open inverted triangle: 3139; closed square: 3143).
  • Figure 4B is a chart showing CD4+ T-cell counts/mm of blood in macaques immunized with ⁇ YNAC during an established SIVmac251 infection (closed circle: 480; open circle: 644; closed inverted triangle: 680; open inverted triangle: 3139; closed square: 3143).
  • Figure 4C illustrates Dryvax-induced skin lesions in macaques immunized with ⁇ YNAC during an established SINmac251 infection.
  • Figure 4D is a chart showing the mean size of skin lesions over time, and a comparison of skin lesions in macaques in Group 2 (open square) (vaccinia-na ⁇ ve) and group 5 (closed square) (immunized with ⁇ YNAC after SIN infection).
  • Figure 5 A is an immunological correlate showing that an inverse immunological correlation between CD4+ T-cell count and the maximal lesion size is significant in vaccinia- na ⁇ ve macaques of Group 1 and Group 2.
  • Figure 5B is an immunological correlate showing that an inverse immunological correlation between CD4+ T-cell count and the maximal lesion size is not significant in vaccinia-experienced macaques.
  • Figure 5C is graph showing the titer of neutralizing antibodies (ID 50 values) that were measured at a single point within the first five weeks from Dryvax challenge in most of the macaques studied.
  • Figure 5D is an immunological correlate showing an inverse correlation between neutralizing antibody titers elicited by Dryvax and time to resolution of skin lesions.
  • highly attenuated vaccinia virus refers to a vaccinia virus that has been genetically altered, often by modern molecular biological methods, e.g. restriction endonuclease and ligase treatment, and rendered less virulent than wild type, typically by deletion of specific genes or by serial passage in a non-natural host cell line or at cold temperatures.
  • These highly attenuated viruses create an abortive infection in mammalian host cells and typically replicate poorly in many mammalian cells, i.e., have a restricted ability to replicate.
  • a "standard vaccinia virus” as used herein refers to a live strain of vaccinia virus that is used to inoculate individuals for immunization against smallpox.
  • the strains are replication-competent in mammalian host cells and can produce large numbers of virus progeny.
  • Such vaccines are live viruses, for example, the Wyeth Dryvax vaccine, traditionally prepared from the skin of calves and the Lister or Elestree (United Kingdom) strain, traditionally prepared on the skin of sheep. These vaccines were generally prepared by harvesting viruses from the skins of the particular animals, although cell culture methods of producing these vaccine strains have recently been developed.
  • an "immunocompromised individual” as used herein refers to a patient with a deficiency in generating either a humoral or cellular immune response.
  • Such patients include individuals with immunodeficiency disease, e.g., HIN infection; individuals undergoing chemotherapy or radiation therapy, or patients with congenital immune defects.
  • the normal range of CD4+ T-cells in a healthy individual is from about 500 to about 1600 cells/mm 3 .
  • the normal range of CD8+ T-cells in a healthy individual is from about 375 to about 1100 cells/mm 3 .
  • Individuals or patients having CD4+ or CD8+ T-cell counts that are lower than the normal range are included within the group of immunocompromised individuals or patients.
  • Efficient CD8 + response refers to the ability of cytotoxic CD8 + T-cells to recognize and kill cells expressing foreign peptides in the context of a major histocompatibility complex (MHC) class I molecule.
  • MHC major histocompatibility complex
  • An "efficient B cell response” refers to the ability of B cells to produce antibodies in response to stimulation by a foreign antigen.
  • Pox viruses are large, enveloped viruses with double-stranded D ⁇ A that is covalently closed at the ends. Pox viruses replicate entirely in the cytoplasm, establishing discrete centers of viral synthesis. Their use as vaccines has been known since the early
  • Vaccinia viruses are orthopoxviruses closesly related to variola and cowpox virus that have an unknown origin and no known natural host. The genes common to vaccinia, variola, and cowpox viruses are greater than 90% identical.
  • the invention provides a method that is directed to immunization of immunocompromised patients to protect against smallpox.
  • the method comprises administering a highly attenuated vaccinia virus, e.g., a genetically altered virus, prior to immunization with a standard smallpox vaccine, i.e., a replication-competent live virus e.g., Dryvax®, Elstree, or Acambis).
  • a highly attenuated vaccinia virus e.g., a genetically altered virus
  • a standard smallpox vaccine i.e., a replication-competent live virus e.g., Dryvax®, Elstree, or Acambis.
  • Dryvax® is a vaccine currently licensed in the United States for smallpox immunization. It is a lyophilized, live- virus preparation of infectious vaccinia virus (Wyeth Laboratories, Inc., Marietta, Pennsylvania). Vaccinia vaccine does not contain smallpox (variola) virus. The vaccine had been prepared from calf lymph with a seed virus derived from the New York City Board of Health (NYCBOH) strain of vaccinia virus and typically has a minimum concentration of 10 pock-formmg units (PFU)/ml. Vaccine was administered by using the multiple-puncture technique with a bifurcated needle. A reformulated vaccine, produced by using cell-culture techniques, is now being developed.
  • vaccinia strains similar to Dryvax® were also used in the world campaign to eradicate smallpox. These include a vaccine derived from the vaccinia strain Lister Elstree and the vaccine "Acambis", which is also derived from the same strain as Dryvax®, the New York City Board of Health strain. Cell-culture adapted vaccines based on these traditional vaccine strains have also been developed or are in the process of being developed.
  • Highly attenuated vaccinia viruses are preferred as a priming vaccine in the methods of the invention.
  • a number ofhighly attenuated poxviruses have been described. A detailed review of this technology is found in US Patent No. 5,863,542 which is incorporated by reference herein.
  • Representative examples of highly attemuated vaccinia viruses include NYVAC and MVA. These viruses were deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852, USA: NYVAC under ATCC accession number VR-2559 on Mar. 6, 1997.
  • NYNAC is a genetically engineered vaccinia virus strain generated by the specific deletion of eighteen open reading frames encoding gene products associated with virulence and host range.
  • ⁇ YVAC is highly attenuated by a number of criteria including: i) decreased virulence after intracerebral inoculation in newborn mice, ii) inocuity in genetically (nu /nu + ) or chemically (cyclophosphamide) immunocompromised mice, iii) failure to cause disseminated infection in immunocompromised mice, iv) lack of significant induration and ulceration on rabbit skin, v) rapid clearance from the site of inoculation, and vi) greatly reduced replication competency on a number of tissue culture cell lines including those of human origin.
  • MVA Modified Vaccinia virus Ankara
  • MVA retains its original immunogenicity and no longer has any virulence and contagiousness for animals and humans.
  • NYVAC abortive infection of human cells occurs, thus providing a safe, yet effective vaccine.
  • a vaccine regimen comprising a priming inoculation with a highly attenuated virus prior to inoculation with a conventional vaccinia virus, e.g., Dryvax, Elstree, etc., provides a safer, i.e., fewer side effects, vaccination regimen in comparison to inoculation with the standard vaccinia virus, yet preserves the protective effects of standard vaccination.
  • This regimen may be used in normal populations, e.g., immunocompetent individuals, but is particularly valuable for immunization of immunocompromised individuals or pregnant women.
  • NYVAC is typically administered as the priming virus.
  • the vaccine regimen can be administered to any human patient in need of such treatment.
  • the vaccine regimen can be administered to patients having CD4+ T- cell counts that are between about 500 and about 1600 cells/mm 3 , and CD8+ T-cell counts that are between about 375 and about 1100 cells/ mm 3 .
  • the vaccine regimen will often be administered to immunocompromised patients. These individuals have a decreased ability to mount an immune response.
  • cancer patients undergoing chemotherapy or radiation therapy individuals with immunodeficiency disorder, e.g., AIDS, and transplant patients who are immmunosuppressed are typically included in this population. Patients with congenital immunodeficiency diseases may also be treated using the methods of the invention.
  • the methods of the invention are appropriate for immunocompromised patients having a reduced CD4+ T-cell count. Such patients are often at increased risk of developing opportunistic infections.
  • the methods of the invention may be used to treat patients that have a CD4+ T-cell count that is less than 350 and which are able to mount an immune response to a smallpox vaccine. Examples of such patients include those having a CD4+ T-cell count that is greater than 250, or greater than 300. Other examples of such immunocompromised patients include those having a CD4+ T-cell count that is between about 200 and about 500, between about 250 and about 500, between about 300 and about 500, or between about 350 and about 500.
  • a vaccine regimen comprising administration of a highly attenuated virus either alone or in combination with a traditional smallpox vaccine may also be used in pregnant women according to the invention. Evaluation of vaccination regimen An effective dose of a highly attenuated vaccinia virus is an amount of vaccine that elicits an immune response, e.g., production of antibodies and/or that decreases the size and time to resolution of Dryvax-induced lesions. Other indicators of an active immune response include CD4+ and CD8+ responses. These responses are all assayed using well known methodology.
  • Antibodies in particular, neutralizing antibodies
  • the presence of neutralizing antibodies may be evaluated in plasma samples using a plaque reduction neutralization test or the techniques described in the examples. Briefly, plasma samples are evaluated for the presence of vaccinia neutralizing antibodies using standard assays such as plaque reduction neutralization tests or an assay described in the examples, which is based on the expression of /3-galactosidase.
  • a recombinant vaccinia virus vSC56 expressing /3-gal under the control of a synthetic early/late (E/L) promoter 19, was used to develop a neutralization assay based on a single-round infection of HeLa cells (CCL-2, American Type Culture Collection, Manassas, Virginia).
  • Each assay includes as a positive control FDA Standard Reference Vaccinia j-tnmunoglobulin.
  • Negative controls included plasma from unvaccinated individuals and albumin.
  • Serial dilutions of each plasma sample are pre-incubated with vSC56 virus and then dispensed into ninety-six-well round bottom plates containing HeLa cells. Plates are incubated for an additional period.
  • Cells are then lysed with detergent and the ⁇ -gal enzymatic activity in each well determined.
  • the ⁇ -gal activity of the tested samples are compared to the level in samples that did not contain plasma from inoculated individuals.
  • Such an assay detects the level of neutralizing antibody in the inoculated patient. Those individuals who demonstrate an immune response to the highly attenuated vaccinia and or the standard vaccinia have antibody levels at least twice above background.
  • CD4+ analysis To assess the effectiveness of the vaccine combination in a recipient or to identify patient populations with CD4+ count of less than 350, a number of assays are available. These assays may also be used to identify patients having a CD4+ T-cell count that is greater than 250 or 300.
  • most laboratories measure absolute CD4 T-cell levels in whole blood by a multi-platform, three-stage process.
  • the CD4 + T-cell number is the product of three laboratory techniques: the white blood cell (WBC) count; the percentage of WBCs that are lymphocytes (differential); and the percentage of lymphocytes that are CD4 + T-cells.
  • WBC white blood cell
  • the last stage in the process of measuring the percentage of CD4 + T-lymphocytes in the whole- blood sample is referred to as "immunophenotyping by flow cytometry.
  • Immunophenotyping refers to the detection of antigenic determinants (which are unique to particular cell types) on the surface of WBCs using antigen-specific monoclonal antibodies that have been labeled with a fluorescent dye or fluorochrome (e.g., phycoerythrin [PE] or fluorescein isothiocyanate [FITC]).
  • a fluorescent dye or fluorochrome e.g., phycoerythrin [PE] or fluorescein isothiocyanate [FITC]
  • the fluorochrome-labeled cells are analyzed by using a flow cytometer, which categorizes individual cells according to size, granularity, fluorochrome, and intensity of fluorescence. Size and granularity, detected by light scattering, characterize the types of WBCs (i.e., granulocytes, monocytes, and lymphocytes).
  • Fluorochrome-labeled antibodies distinguish populations and subpopulations of WBCs.
  • CD4 + cells are commercially available.
  • Becton Dickenson's FACSCount System automatically measure absolutes CD4 + , CD8 + , and CD3 + T lymphocytes. It is a self-contained system, incorporating instrument, reagents, and controls. A successful increase of CD4 + cell counts would be a 2X or higher increase in the number of CD4 + cells.
  • CD8 T-cell responses may be measured, for example, by using tetramer staining of fresh or cultured PBMC, ELISPOT assays or by using functional cytotoxicity assays, which are well-known to those of skill in the art.
  • a functional cytotoxicity assay can be performed as follows. Briefly, peripheral blood lymphocytes are cultured with a peptide epitope, such as an epitope that overlaps from one early protein and one late region of variola major. Following three days of culture, the medium is supplemented with human IL-2 and the cultures are maintained for four additional days.
  • PBLs are centrifuged over Ficoll- Hypaque and assessed as effector cells in a standard 51 Cr-release assay using U-bottomed micro titer plates containing about 10 4 target cells with varying effector cell concentrations. All cells are assayed twice. Autologous B lymphoblastoid cell lines are used as target cells and are loaded with peptide by incubation overnight during 51 Cr labeling. Specific release is calculated in the following manner: (experimental release-spontaneous release)/(maximum release-spontaneous release) x 100. Spontaneous release is generally less than 20% of maximal release with detergent (2% Triton X-100) in all assays. A successful CD8 + response occurs when the induced cytolytic activity is above 10% of controls.
  • CD8 + responses provides direct quantification of antigen- specific T cells by staining with Fluorescein-labeled HLA tetrameric complexes (Airman, J. D. et al, Proc. Natl Acad. Set USA 90:10330, 1993; Airman, J. D. et al, Science 274:94, 1996).
  • Other assays include staining for intracellular lymphokines, and ⁇ -interferon release assays or ELISPOT assays. Tetramer staining, intracellular lymphokine staining and
  • ELISPOT assays all are sensitive measures of T cell response (Lalvani, A. et al, J. Exp. Med. 186:859, 1997; Dunbar, P. R. et al, Curr. Biol. 8:413, 1998; Murali-Krislina, K. et al, Immunity 8:177, 1998).
  • Vaccine compositions e.g., compositions containing the standard smallpox virus can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration.
  • the vaccine may be delivered in a physiologically compatible solution such as sterile PBS in a volume of, e.g., one ml.
  • the vaccines may also be lyophilized prior to delivery. As well known to those in the art, the dose may be proportional to weight.
  • compositions included in the vaccine regimen can be administered alone, or can be co-administered or sequentially administered with other immunological, antigenic, vaccine, or therapeutic compositions.
  • These include adjuvants, and chemical or biological agent given in combination with, or recombinantly fused to, an antigen to enhance immunogenicity of the antigen.
  • Such other compositions can also include biological response modifiers (e.g., cytokines or co-stimulating molecules).
  • biological response modifiers include IL-2, IL-7, and CD40 ligand.
  • adjuvants examples include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum). Again, co-administration is performed by taking into consideration such known factors as the age, sex, weight, and condition of the particular patient, and, the route of administration.
  • the vaccines can additionally be complexed with other components such as peptides, polypeptides and carbohydrates for delivery.
  • Vaccines can be delivered via a variety of routes. Typical delivery routes include intradermal administration, but other routes, e.g., intramuscular, subcutaneous, epicutaneous or parenteral routes may also be employed.
  • the vaccines can also be formulated for administration via mucosal passage, e.g., nasal passages or administered intrabucally.
  • Formulations suitable for nasal administration, wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is administered in the manner in which snuff is taken, i. e. , by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer include aqueous or oily solutions of the active ingredient.
  • vaccine compositions of use for the invention include liquid preparations, for orifice, e.g., oral, nasal, anal, vaginal, etc. administration, such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions, h such compositions the vaccinia virus can be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like.
  • the highly attenuated virus is introduced into a human vaccinee by standard methods for vaccination of live vaccines.
  • a live vaccine of the invention can be administered at, for example, about 10 4 -10 8 organisms/dose, or 10 6 to 10 9 pfuper dose.
  • Actual dosages of such a vaccine can be readily determined by one of ordinary skill in the field of vaccine technology.
  • a highly attenuated vaccinia virus is typically administered in an amount of about 10 4 to about 10° pfu per inoculation; often about 10 5 pfu to about 10 s pfu.
  • Higher dosages such as about 10 4 pfu to about 10 1 pfu, e.g., about 10 5 pfu to about 10 9 pfu, or about 10 pfu to about 10 pfu, can also be employed.
  • a NYVAC- vaccine can be inoculated by a route, e.g., an intramuscular route at a dose of about 10 pfu or more per inoculation, for a patient of 170 pounds.
  • the highly attenuated virus may be administered more than once, as determined by the response of the particular patient, hi addition, a patient may receive an inoculation of one highly attenuated virus, e.g., NYVAC, followed by an inoculation of a different highly attenuated virus, e.g., MVA.
  • the amount of time between multiple administrations of the highly attenuated virus primer can be determined using standards known to those in the art, taking into account the medical status of the patient.
  • Standard smallpox vaccines are typically administered to the skin using a bifurcated needle.
  • the skin over the insertion of the deltoid muscle or the posterior aspect of the arm over the triceps are generally the preferred sites for smallpox vaccination.
  • the multiple- puncture technique uses a pre-sterilized bifurcated needle that is inserted vertically into the vaccine vial, causing a droplet of vaccine to adhere between the prongs. The droplet contains the recommended dosage of the vaccine.
  • Multiple punctures, typically 15, are then performed to introduce the vaccine into the skin.
  • the standard smallpox vaccine is usually administered at least 1 week, preferably 2 weeks after administration of the highly attenuated vaccinia virus.
  • Recipients of standard potency vaccinia vaccine usually received a controlled percutaneous dose of about 2.5 x 10 5 pfu. Smaller doses, about 10 4 pfu or larger doses, e.g., 10 , may also be used if required.
  • the concentration of virus provided in currently available preparations is typically about 10 pfu/ml.
  • the recipient receives at least one dose of the standard vaccine, although multiple doses maybe administered if judged necessary.
  • Described in this example are studies using macaques with severe depletion of CD4+ T cells consequent to simian immunodeficiency virus (SIN) or simian/human immunodeficiency virus (SHIV) 89.6 infection to test the ability of prior immunization with the highly attenuated poxviruses ⁇ YVAC and modified vaccinia virus Ankara (MVA) to protect against complications of Dryvax vaccination.
  • SIN simian immunodeficiency virus
  • SHIV simian/human immunodeficiency virus
  • Table 1 Size of skin lesions in vaccinia-na ⁇ ve or -experienced immune-competent and vaccinia-na ⁇ ve immune-compromised macaques following Dryvax immunization.
  • infection with the SIVmac251 viral strain which also causes chronic viremia, is characterized by slow progressive CD4+ T-cell loss and acquired immune deficiency syndrome (AIDS) development with two to three years from infection (Desrosiers, R. C.
  • macaque 20M which received ⁇ YNAC-SIV, in the amount of 10 pfu each except macaques 17697 and 17294, which received 10 7 pfu per inoculation.
  • Macaque 17294 which had no detectable CD4+ T-cells at the time of immunization, developed an enlarged lesion with a maximal size of 1.1 cm but, nevertheless, by day 26 the lesion was resolved and progressive vaccinia was resolved (Figs. 3G and 3H). Similarly, three immuno-deficient macaques previously immunized with MNA experienced resolution of the Dryvax lesions within three weeks (Figs. 31 and 3J).
  • ⁇ YNAC vaccination during an established S ⁇ Vmac251 infection decreases the size and time to resolution of Dryvax lesions.
  • CD4+ T-cell counts in HIN- 1 -infected individuals have been considered the hallmark of disease progression and a predictive clinical parameter for the occurrence of opportunistic infections.
  • a correlation analysis was performed using the CD4+ T-cell count at the time of challenge and the size of the maximal lesion observed.
  • vaccinia-na ⁇ ve macaques from groups 1 and 2 which had a wide range of CD4+ T-cell counts (Figs.
  • CD4+ T-cells likely play an important role in containing Dryvax replication and that the CD4+ T-cell level may predict an adverse outcome of Dryvax vaccination in vaccinia-na ⁇ ve individuals with acquired immune deficiency.
  • the titer of neutralizing antibodies elicited by Dryvax inversely correlates with time to lesion resolution.
  • Plasma virus level, and CD4+ T-cell count are summarized in Figs. 2A-2D. Most of the macaques in this group had severe depletion of CD4+ T-cells and their mean CD4+ T- cells/mm 3 was 121 ⁇ 52.
  • groups 3, 4, and 5 no single group had a significantly different CD4+ T-cell count from any of the others.
  • the area of the smallpox vaccination plaques was measured using a "region measurements" tool of Metamorph (Universal Imaging Corporation, Downingtown, Pennsylvania). Two-group comparisons of lesion sizes, times to resolution of lesions, and CD4+ T-cell counts were made using the exact Wilcoxon rank sum test, and correlations between them were tested by the Spearman rank correlation method (StatXact Version 4.0.1 , Cytel Software Corporation, Cambridge, Massachusetts). A CD4+ T-cell count of 0 was assigned the value 10 when plotted on a logarithmic scale (Fig. 5B).
  • Neutralizing antibodies ff-galactosidase-based vaccinia neutralization assay. Plasma samples from representative monkeys in groups 1-5 were collected immediately prior to Dryvax challenge (day 0) and at post-challenge times (ranging from seven to forty days for different monkeys). All plasma samples were heat-inactivated (56 °C for thirty minutes) and were evaluated for the presence of vaccinia neutralizing antibodies using a novel assay based on expression of a reporter gene, /5-galactosidase (/3-gal) (Manischewitz, J., King, L.R., Bleckwenn, N.
  • a recombinant vaccinia virus vSC56 expressing /3-gal under the control of a synthetic early/late (E/L) promoter (Chakrabarti et al, Biotechniques 23: 1094-1097, 1997), was used to develop a neutralization assay based on a single-round infection of HeLa cells (CCL-2, American Type Culture Collection, Manassas, Virginia). This is a rapid (twenty- four hour), high throughput assay that was shown to have similar sensitivity to the classical plaque reduction neutralization tests (PRNT) (Manischewitz, J., King, L.R., Bleckwenn, N.
  • PRNT plaque reduction neutralization tests
  • Each assay includes as a positive control FDA Standard Reference Vaccinia Immunoglobulin (VIG) obtained from Dynport vaccine company (Frederick, Maryland) and vialed at the Center for Biologies Evaluation and Research. Negative controls included plasma from unvaccinated children and albumin.
  • VOG Standard Reference Vaccinia Immunoglobulin
  • Each plate included a /3-gal standard curve using a recombinant /3-gal enzyme (Roche Diagnostics Corporation, Indianapolis, Indiana). Chlorophenol red beta-D-galactopyranoside monosodium salt (CPRG) substrate (Roche Diagnostics) was added to all wells for thirty minutes at room temperature in the dark, and the enzymatic reaction was stopped with 1M Na 2 CO solution. Optical density (OD) was determined at 575 nm by an enzyme-linked immunosorbent assay (ELISA) reader. OD readings were transferred to Microsoft Excel for further analysis. The /3-gal standard curves were used to convert OD values into / 3-gal activity per experimental or control group (in mU/ml).
  • CPRG Chlorophenol red beta-D-galactopyranoside monosodium salt
  • HlV-infected individual with a CD4+ count of less than 350 is immunized against smallpox infection using the method of the invention.
  • a dose of about 5 x 10 8 pfu of NYVAC is administered to a 170 lb male with a CD4+ count of less than 350.
  • the patient's serum is monitored for the presence of antibodies two weeks following administration. If antibodies are detected, the patient is then administered a dose of about 10 5 pfu of Dryvax. The results show that neutralizing antibodies are present in the patient serum.

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Abstract

La présente invention concerne un schéma posologique amélioré pour l'administration d'un vaccin antivariolique à une population immunodéprimée ou enceinte.
PCT/US2003/035499 2002-11-07 2003-11-06 Schema posologique pour vaccin antivariolique WO2004043490A1 (fr)

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WO2003088994A2 (fr) * 2002-04-19 2003-10-30 Bavarian Nordic A/S Virus de la vaccine ankara modifiee pour la vaccination des nouveau-nes

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003088994A2 (fr) * 2002-04-19 2003-10-30 Bavarian Nordic A/S Virus de la vaccine ankara modifiee pour la vaccination des nouveau-nes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BELYAKOV I.M. ET AL.: "Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses", PNAS, vol. 100, no. 16, 5 August 2003 (2003-08-05), pages 9458 - 9463, XP002975724 *
EDGHILL-SMITH Y. ET AL.: "Modeling a safer smallpox vaccination regimen, for human immunodeficiency virus type 1-infected patients, in immunocompromised macaques", JOURNAL OF INFECTIOUS DISEASES, vol. 188, 15 October 2003 (2003-10-15), pages 1181 - 1191, XP002975723 *
HENDERSON D.A. ET AL.: "Smallpox as a biological weapon - medical and public health management", JAMA, vol. 281, no. 22, 9 June 1999 (1999-06-09), pages 2127 - 2137, XP002951395 *
M.E.: "In search of a kinder, gentler vaccine", SCIENCE, vol. 296, 31 May 2002 (2002-05-31), pages 1594, XP002975024 *

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